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THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 


Editorial  Board 

DANIEL  L.  ALKON,  National  Institutes  of  Health  and    MICHAEL  G.  O'RAND,  Laboratories  for  Cell  Biology, 

Marine  Biological  Laboratory  University  of  North  Carolina  at  Chapel  Hill 

ROBERT  B.  BARLOW,  JR.,  Syracuse  University  **"*«  S-  QUATRANO,  Oregon  State  University  at 

Corvallis 

STEPHEN  C.  BROWN,  State  University  of  New  York    JQEL  L  RoSENBAUM)  Yale  University 

at  Albany 

DOROTHY  M.  SKINNER,  Oak  Ridge  National 
DAVID  H.  EVANS,  University  of  Florida  Laboratory 

HARLYN  O.  HALVORSON,  Brandeis  University  JoHN  D-  STRANDBERG,  Johns  Hopkins  University 

SAMUEL  S.  KOIDE,  The  Population  Council,  J-  RICHARD  WHITTAKER,  Boston  University 

Rockefeller  University        Marine  Program  and  Marine  Biological  Laboratory 

E.  O.  WILSON,  Harvard  University 
FRANK  J.  LONGO,  University  of  Iowa 

GEORGE  M.  WOODWELL,  Ecosystems  Center,  Marine 
GEORGE  O.  MACKIE,  University  of  Victoria  Biological  Laboratory 


Editor:  CHARLES  B.  METZ,  University  of  Miami 


VOLUME  163 

JULY  TO  DECEMBER,  1982 


Printed  and  Issued  by 
LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 
LANCASTER,  PA. 


The  BIOLOGICAL  BULLETIN  is  issued  six  times  a  year  at  the 
Lancaster  Press,  Inc.,  Prince  and  Lemon  Streets,  Lancaster,  Penn- 
sylvania. 

Subscriptions  and  similar  matter  should  be  addressed  to  The 
Biological  Bulletin,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts.  Single  numbers,  $10.00.  Subscription  per  volume 
(three  issues),  $27.00  ($54.00  per  year  for  six  issues). 

Communications  relative  to  manuscripts  should  be  sent  to  Dr. 
Charles  B.  Metz,  Marine  Biological  Laboratory,  Woods  Hole,  Mas- 
sachusetts 02543  between  June  1  and  September  1,  and  to  Dr. 
Charles  B.  Metz,  Institute  For  Molecular  and  Cellular  Evolution, 
University  of  Miami,  521  Anastasia,  Coral  Gables,  Florida  33134 
during  the  remainder  of  the  year. 


THE  BIOLOGICAL  BULLETIN  (ISSN  0006-3185) 
Second-class-postage  paid  at  Woods  Hole,  Mass.,  and  additional  mailing  offices. 


LANCASTER  PRESS,  INC..  LANCASTER.  PA. 


CONTENTS 

No.  1,  AUGUST  1982 

ANNUAL  REPORT  OF  THE  MARINE  BIOLOGICAL  LABORATORY    1 

Invited  article: 

EVANS,  DAVID  H.,  J.  B.  CLAIBORNE,  LINDA  FARMER,  CHARLES  MALLERY, 

AND  EDWARD  J.  KRASNY,  JR. 

Fish  gill  ionic  transport:  methods  and  models     108 

COPELAND,  D.  EUGENE 

The  anatomy  and  fine  structure  of  the  eye  in  fish.  VI  ciliary  type  tissue 

in  nine  species  of  teleosts    131 

DEVINE,  DANA  V.,  AND  JELLE  ATEMA 

Function  of  chemoreceptor  organs  in  spatial  orientation  of  the  lobster, 
Homarus  americanus:  differences  and  overlap    144 

FINGER,  THOMAS  E. 

Somatotopy  in  the  representation  of  the  pectoral  fin  and  free  fin  rays  in 

the  spinal  cord  of  the  sea  robin,  Prionotus  carolimis    154 

GLEESON,  RICHARD  A. 

Morphological  and  behavioral  identification  of  the  sensory  structures 
mediating  pheromone  reception  in  the  blue  crab,  Callinectes  sapidus    162 

JEBRAM,  DIETHARDT,  AND  BETTY  EVERITT 

New  Victorellids  (Bryozoa,  Ctenostomata)  from  North  America:  the  use 

of  parallel  cultures  in  Bryozoan  taxonomy    172 

LAWN,  I.  D.,  AND  D.  M.  Ross 

The  release  of  the  pedal  disk  in  an  undescribed  species  of  Tealia  (An- 
thozoa:  Actiniaria) 188 

MALLATT,  JON 

Pumping  rates  and  particle  retention  efficiencies  of  the  larval  lamprey, 

an  unusual  suspension  feeder   197 

POHLE,  GERHARD,  AND  MALCOLM  TELFORD 

Post-larval  growth  of  Dissodactylus  primitivus  Bouvier,  1917  (Brachyura: 
Pinnotheridae)  under  laboratory  conditions    211 

REED-MILLER,  CHARLENE,  AND  MICHAEL  J.  GREENBERG 

The  ciliary  junctions  of  scallop  gills:  the  effects  of  cytochalasins  and  con- 
canavalin  A    225 

SlEBENALLER,  JOSEPH  F.,  GEORGE  N.  SOMERO,  AND  RICHARD  L.  HAEDRICH 

Biochemical  characteristics  of  macrourid  fishes  differing  in  their  depths 

of  distribution    240 

No.  2,  OCTOBER  1982 

CHRISTY,  JOHN  H. 

Adaptive  significance  of  semilunar  cycles  of  larval  release  in  fiddler  crabs 
(genus  Uca):  test  of  an  hypothesis    251 

EMLET,  RICHARD  B. 

Echinoderm  calcite:  a  mechanical  analysis  from  larval  spicules     264 

ESCALONA  DE  MOTTA,  GLADYS,  DAVID  S.  SMITH,  MARILYN  CAYER,  AND 

JOSE  DEL  CASTILLO 

Mechanism  of  the  excitation-contraction  uncoupling  of  frog  skeletal  mus- 
cle by  formamide     276 

FORWARD,  R.  B.,  JR.,  K.  LOHMANN,  AND  T.  W.  CRONIN 

Rhythms  in  larval  release  by  an  estuarine  crab  (Rhithropanopeus  harrisii )    287 

iii 


IV 


CONTENTS 


HOPKINS,  PENNY  M. 

Growth  and  regeneration  patterns  in  the  fiddler  crab,   Uca  pugilator    301 

MARTIN,  VICKI  J.,  AND  FU-SHIANG  CHIA 

Fine  structure  of  a  scyphozoan  planula,  Cassiopeia  xamachana 320 

O'CONNOR,  KATHLEEN,  PHILIP  J.  STEPHENS,  AND  JOHN  M.  LEFEROVICH 
Regional  distribution  of  muscle  fiber  types  in  the  asymmetric  claws  of 
Californian  snapping  shrimp     329 

SCHUEL,  HERBERT,  PRAMILA  DANDEKAR,  AND  REGINA  SCHUEL 

Urea  parthenogenetically  activates  the  cortical  reaction  and  elongation 

of  microvilli  in  eggs  of  the  sea  urchin,  Strongylocentrotus  purpuratus    337 

WATTS,  STEPHEN  A.,  R.  E.  SCHEIBLING,  ADAM  G.  MARSH,  AND  JAMES  B. 

McCLINTOCK 

Effect  of  temperature  and  salinity  on  larval  development  of  sibling  species 

of    Echinaster    (Echinodermata:    Asteroidea)    and    their    hybrids  348 

ABSTRACTS  OF  PAPERS  PRESENTED  AT  THE  GENERAL  SCIENTIFIC  MEETINGS 

OF  THE  MARINE  BIOLOGICAL  LABORATORY 

Actin,  microtubules,  etc 355 

Ecology 362 

Fertilization  and  development    371 

Neurobiology   379 

Parasitology  and  pathology  39 1 

Photoreceptors  394 

Physiology  and  biophysics     397 

No.  3,  DECEMBER  1982 

Invited  article: 

PIERCE,  SIDNEY  K. 

Invertebrate  cell  volume  control  mechanisms:  a  coordinated  use  of  in- 
tracellular  amino  acids  and  inorganic  ions  as  osmotic  solute 405 

DUNHAM,  PHILIP,  LEONARD  NELSON,  LESLIE  VOSSHALL,  AND  GERALD 

WEISSMAN 

Effects  of  enzymatic  and  nonenzymatic  proteins  on  Arbacia  spermatozoa: 
reactivation  of  aged  sperm  and  the  induction  of  polyspermy  420 

HENDLER,  GORDON 

An  echinoderm  vitellaria  with  a  bilateral  larval  skeleton:  evidence  for  the 
evolution  of  ophiuroid  vitellariae  from  ophioplutei  431 

KANUNGO,  K. 

In  vitro  studies  on  the  effects  of  cell-free  coelomic  fluid,  calcium,  and/or 
magnesium  on  clumping  of  coelomocytes  of  the  sea  star  Asterias  forbesi 
(Echinodermata:  Asteroidea)  438 

LONGO,  FRANK  J.,  AND  ALLEN  W.  SCHUETZ 

Male  pronuclear  development  in  starfish  oocytes  treated  with  1-meth- 
yladenine  453 

LONGO,  FRANK  J.,  FREDERICK  So,  AND  ALLEN  W.  SCHUETZ 

Meiotic  maturation  and  the  cortical  granule  reaction  in  starfish  eggs    465 

MAURER,  DON,  AND  ROLAND  L.  WIGLEY 

Distribution  and  ecology  of  mysids  in  Cape  Cod  Bay,  Massachusetts    477 

NELSON,  LEONARD 

Membrane-stabilizing  and  calcium-blocking  agents  affect  Arbacia  sperm 
motility  492 

WARREN,  MARY  KIM,  AND  SIDNEY  K.  PIERCE 

Two  cell  volume  regulatory  systems  in  the  Limulus  myocardium:  an 
interaction  of  ions  and  quaternary  ammonium  compounds  504 

INDEX  TO  VOLUME  163  517 


Volume  163  Number  1 

,  -  -  .  s 

• 


THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 

Editorial  Board 

DANIEL  L.  ALKON,  National  Institutes  of  Health  and     MICHAEL  G.  O'RAND,  Laboratories  for  Cell  Biology, 
Marine  Biological  Laboratory  University  of  North  Carolina  at  Chapel  Hill 

ROBERT  B.  BARLOW,  JR.,  Syracuse  University  RALPH  S.  QUATRANO,  Oregon  State  University  at 

Corvallis 

STEPHEN  C.  BROWN,  State  University  of  New  York    JQEL  L  RosENBAUM)  Yale  University 

at  Albany 

DOROTHY  M.  SKINNER,  Oak  Ridge  National 
DAVID  H.  EVANS,  University  of  Florida  Laboratory 

HARLYN  O.  HALVORSON,  Brandeis  University  JOHN  D.  STRANDBERG,  Johns  Hopkins  University 

J.  RICHARD  WHITTAKER,  Boston  University 
SAMUEL  S.  KOIDE,  The  Populate  Council  d  Mafine  Biol     ical  Laboratory 

Rockefeller  University 

E.  O.  WILSON,  Harvard  University 
FRANK  J.  LONGO,  University  of  Iowa  ...    . 

GEORGE  M.  WOODWELL,  Ecosystems  Center,  Marine 

GEORGE  O.  MACKIE,  University  of  Victoria  Biological  Laboratory 

Editor:  CHARLES  B.  METZ,  University  of  Miami 


AUGUST,  1982 


Printed  and  Issued  by 
LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 
LANCASTER,  PA. 


THE  BIOLOGICAL  BULLETIN 

/  V"  '  /.   a  •sSHT1    ->  '^ s' 

THE  BIOLOGICAL  BULLETIN  is  published  six  times  a  year  by  the  Marine  Biological  Laboratory, 
MBL  Street,  Woods  Hole,  Massachusetts  02543. 

Subscriptions  and  similar  matter  should  be  addressed  to  THE  BIOLOGICAL  BULLETIN,  Marine  Bi- 
ological Laboratory,  Woods  Hole,  Massachusetts.  Single  numbers,  $10.00.  Subscription  per  volume 
(three  issues),  $27.00  (this  is  $54.00  per  year  for  six  issues). 

Communications  relative  to  manuscripts  should  be  sent  to  Dr.  Charles  B.  Metz,  Editor,  or  Helen 
Lang,  Assistant  Editor,  at  the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts  02543  between 
June  1  and  September  1,  and  at/the  Institute  For  Molecular  and  Cellular  Evolution,  University  of 
Miami,  521  Anastasia,  Coral  Gables,  Florida  33134  during  the  remainder  of  the  year. 


Copyright  ©  1982,  by  the  Marine  Biological  Laboratory 

Second-class  postage  paid  at  Woods  Hole,  Mass.,  and  additional  mailing  offices. 

ISSN  0006-3185 


g  ,";  INSTRUCTIONS  TO  AUTHORS  gj^j 

THE  BIOLOGICAL  BULLETIN  accepts  original  research  reports  of  intermediate  length  on  a  variety 
of  subjects  of  biological  interest.  In  general,  these  papers  are  either  of  particular  interest  to  workers  at 
the  Marine  Biological  Laboratory,  or  of  outstanding  general  significance  to  a  large  number  of  biologists 
throughout  the  world.  Normally,  review  papers  (except  for  a  limited  number  of  solicited  review  papers 
which  may  be  accepted  after  formal  refereeing),  very  short  papers  (less  than  five  printed  pages),  pre- 
liminary notes,  and  papers  which  describe  only  a  new  technique  or  method  without  presenting  substantial 
quantities  of  data  resulting  from  the  use  of  the  new  method  cannot  be  accepted  for  publication.  A  paper 
will  usually  appear  within  four  months  of  the  date  of  its  acceptance. 

The  Editorial  Board  requests  that  manuscripts  conform  to  the  requirements  set  below;  those  manu- 
scripts which  do  not  conform  will  be  returned  to  authors  for  correction  before  review. 

'     \  M  1- I 

1.  Manuscripts.     Manuscripts,  including  figures,  should  be  submitted  in  triplicate.  (Xerox  copies 
of  photographs  are  not  acceptable  for  review  purposes.)  The  original  manuscript  must  be  typed  in  double 
spacing  (including  figure  legends,  footnotes,  bibliography,  etc.)  on  one  side  of  16-  or  20-lb.  bond  paper, 
8Vi  by  11  inches.  Manuscripts  should  be  proofread  carefully  and  errors  corrected  legibly  in  black  ink. 
Pages  should  be  numbered  consecutively.  Margins  on  all  sides  should  be  at  least  1  inch  (2.5  cm). 
Manuscripts  should  conform  to  the  Council  of  Biology  Editors  Style  Manual,  4th  Edition  (Council  of 
Biology  Editors,  1978)  and  to  American  spelling.  Unusual  abbreviations  should  be  kept  to  a  minimum 
and  should  be  spelled  out  on  first  reference  as  well  as  defined  in  a  footnote  on  the  title  page.  Manuscripts 
should  be  divided  into  the  following  components:  Title  page,  Abstract  (of  no  more  than  200  words), 
Introduction,  Materials  and  Methods,  Results,  Discussion,  Acknowledgments,  Literature  Cited,  Tables, 
and  Figure  Legends.  In  addition,  authors  should  supply  a  list  of  words  and  phrases  under  which  the 
article  should  be  indexed. 

2.  Figures.     Figures  should  be  no  larger  than  8'/2  by  1 1  inches.  The  dimensions  of  the  printed  page, 
5  by  73/4  inches,  should  be  kept  in  mind  in  preparing  figures  for  publication.  We  recommend  that  figures 
be  about  1  '/i  times  the  linear  dimensions  of  the  final  printing  desired,  and  that  the  ratio  of  the  largest 
to  the  smallest  letter  or  number  and  of  the  thickest  to  the  thinnest  line  not  exceed  1:1.5.  Explanatory 
matter  generally  should  be  included  in  legends,  although  axes  should  always  be  identified  on  the  illus- 
tration itself.  Figures  should  be  prepared  for  reproduction  as  either  line  cuts  or  halftones.  Figures  to  be 
reproduced  as  line  cuts  should  be  unmounted  glossy  photographic  reproductions  or  drawn  in  black  ink 
on  white  paper,  good-quality  tracing  cloth  or  plastic,  or  blue-lined  coordinate  paper.  Those  to  be  re- 
produced as  halftones  should  be  mounted  on  board,  with  both  designating  numbers  or  letters  and  scale 
bars  affixed  directly  to  the  figures.  All  figures  should  be  numbered  in  consecutive  order,  with  no  distinction 
between  text  and  plate  figures.  The  author's  name  and  an  arrow  indicating  orientation  should  appear 
on  the  reverse  side  of  all  figures. 

•V-  )J.^jt  -  ,-."  ,'•   ; 

3.  Tables,  footnotes,  figure  legends,  etc.     Authors  should  follow  the  style  in  a  recent  issue  of  The 

Biological  Bulletin  in  preparing  table  headings,  figure  legends,  and  the  like.  Because  of  the  high  cost 
of  setting  tabular  material  in  type,  authors  are  asked  to  limit  such  material  as  much  as  possible.  Tables, 

Continued  on  Cover  Three 


THE  MARINE  BIOLOGICAL  LABORATORY 

EIGHTY-FOURTH  REPORT,  FOR  THE  YEAR  1981— NINETY-FOURTH  YEAR 

I.     TRUSTEES  AND  STANDING  COMMITTEES    1 

II.     MEMBERS  OF  THE  CORPORATION  5 

1 .  LIFE  MEMBERS    5 

2.  REGULAR  MEMBERS 

3.  ASSOCIATE  MEMBERS 25 

III.  CERTIFICATE  OF  ORGANIZATION   28 

IV.  ARTICLES  OF  AMENDMENT  29 

V.     BYLAWS  30 

VI.     REPORT  OF  THE  DIRECTOR   34 

VII.     REPORT  OF  THE  CONTROLLER    48 

VIII.     REPORT  OF  THE  TREASURER 49 

IX.     REPORT  OF  THE  LIBRARIAN    62 

X.     EDUCATIONAL  PROGRAMS    62 

1.  SUMMER  62 

2.  JANUARY    71 

3.  SHORT  COURSES   75 

XI.     RESEARCH  AND  TRAINING  PROGRAMS   81 

1.  SUMMER  81 

2.  YEAR-ROUND  91 
XII.     HONORS    97 

XIII.  INSTITUTIONS  REPRESENTED    101 

XIV.  LABORATORY  SUPPORT  STAFF                                             105 


I.  TRUSTEES 

Including  Action  of  the  1981  Annual  Meeting 

OFFICERS 

PROSSER  GIFFORD,  Chairman  of  the  Board  of  Trustees,  Woodrow  Wilson  International 
Center  for  Scholars,  Smithsonian  Building,  Washington,  DC  20560 

DENIS  M.  ROBINSON,  Honorary  Chairman  of  the  Board  of  Trustees,  High  Voltage  Engi- 
neering Corporation,  Burlington,  MA  01803 

ROBERT  MAINER,  Treasurer,  The  Boston  Company,  One  Boston  Place,  Boston,  MA  02106 


Copyright  ©  1982,  by  the  Marine  Biological  Laboratory 

Library  of  Congress  Card  No.  A38-518 

(ISSN  0006-3185) 


MARINE  BIOLOGICAL  LABORATORY 

PAUL  R.  GROSS,  President  of  the  Corporation  and  Director  of  the  Laboratory,  Marine 
Biological  Laboratory,  Woods  Hole,  MA  02543 

EMERITI 

PHILIP  B.  ARMSTRONG,  51  Elliot  Place,  Rutherford,  NJ  (deceased  1/82) 

FRANK  A.  BROWN,  JR.,  Marine  Biological  Laboratory 

JOHN  B.  BUCK,  National  Institutes  of  Health 

AURIN  CHASE,  Princeton  University 

ANTHONY  C.  CLEMENT,  Emory  University 

KENNETH  S.  COLE,  San  Diego,  CA 

ARTHUR  L.  COLWIN,  University  of  Miami 

LAURA  H.  COLWIN,  University  of  Miami 

D.  EUGENE  COPELAND,  Marine  Biological  Laboratory 

SEARS  CROWELL,  Indiana  University 

HARRY  GRUNDFEST,  Columbia  University 

TERU  HAYASHI,  Miami,  FL 

HOPE  HIBBARD,  Oberlin  College 

LEWIS  KLEINHOLZ,  Reed  College 

MAURICE  KRAHL,  Tucson,  AZ 

DOUGLAS  MARSLAND,  Cockeysville,  MD 

HAROLD  H.  PLOUGH,  Amherst,  MA 

C.  LADD  PROSSER,  University  of  Illinois 

JOHN  S.  RANKIN,  Ashford,  CT 

A.  C.  REDFIELD,  Woods  Hole,  MA 

MERYL  ROSE,  Waquoit,  MA 

MARY  SEARS,  Woods  Hole,  MA 

CARL  C.  SPEIDEL,  University  of  Virginia  (no  mailings) 

H.  BURR  STEINBACH,  Woods  Hole,  MA  (deceased  12/81) 

ALBERT  SZENT-GYORGYI,  Marine  Biological  Laboratory 

W.  RANDOLPH  TAYLOR,  University  of  Michigan 

GEORGE  WALD,  Harvard  University 

CLASS  OF  1985 

ROBERT  W.  ASHTON,  Gaston  Snow  Beekman  and  Bogue,  New  York,  NY 

HARLYN  O.  HALVORSON,  Brandeis  University 

JOHN  G.  HILDEBRAND,  Columbia  University 

THOMAS  J.  HYNES,  JR.,  Meredith  &  Grew,  Inc.,  Boston,  MA 

SHINYA  INOUE,  Marine  Biological  Laboratory 

RICHARD  P.  MELLON,  Richard  King  Mellon  Foundation,  Laughlintown,  PA 

JOHN  W.  MOORE,  Duke  University 

W.  D.  RUSSELL-HUNTER,  Syracuse  University 

EVELYN  SPIEGEL,  Dartmouth  College 

CLASS  OF  1984 

CLAY  ARMSTRONG,  University  of  Pennsylvania 

ROBERT  B.  BARLOW,  JR.,  Syracuse  University 

JUDITH  GRASSLE,  Marine  Biological  Laboratory 

HOLGER  JANNASCH,  Woods  Hole  Oceanographic  Institution 

BENJAMIN  KAMINER,  Boston  University 

BRIAN  SALZBERG,  University  of  Pennsylvania 

W.  NICHOLAS  THORNDIKE,  Boston,  MA 

RICHARD  W.  YOUNG,  Cambridge,  MA 


TRUSTEES  AND  STANDING  COMMITTEES 

CLASS  OF  1983 

NINA  ALLEN,  Dartmouth  College 

HAYS  CLARK,  Avon  Products,  Incorporated 

DENNIS  FLANAGAN,  Scientific  American 

WILLIAM  T.  GOLDEN,  New  York,  NY 

PHILIP  GRANT,  University  of  Oregon 

JOEL  ROSENBAUM,  Yale  University 

ANN  STUART,  University  of  North  Carolina 

ANDREW  SZENT-GYORGYI,  Brandeis  University 

KENSAL  VAN  HOLDE,  Oregon  State  University 

CLASS  OF  1982 

EVERETT  ANDERSON,  Harvard  Medical  School 

GEORGE  H.  A.  CLOWES,  JR.,  The  Cancer  Research  Institute,  Boston,  MA 

ELLEN  R.  GRASS,  The  Grass  Foundation 

JOHN  P.  KENDALL,  Boston,  MA 

EDWARD  A.  KRAVITZ,  Harvard  Medical  School 

HANS  LAUFER,  University  of  Connecticut 

MARJORIE  R.  STETTEN,  National  Institutes  of  Health 

WALTER  S.  VINCENT,  University  of  Delaware 

J.  RICHARD  WHITTAKER,  Marine  Biological  Laboratory 


STANDING  COMMITTEES 

EXECUTIVE  COMMITTEE  OF  THE  BOARD  OF  TRUSTEES 

PROSSER  GIFFORD*  NINA  ALLEN,  1983 

PAUL  GROSS*  ANDREW  SZENT-GYORGYI,  1983 

ROBERT  MAINER*  JOEL  ROSENBAUM,  1982 

JOHN  HILDEBRAND,  1984  MARJORIE  STETTEN,  1982 
BENJAMIN  KAMINER,  1984 

BUDGET  COMMITTEE 

JOHN  M.  ARNOLD,  Chairman  ROBERT  MAINER* 

GEORGE  H.  A.  CLOWES,  JR.  JEROME  SCHIFF 

PAUL  GROSS*  HOMER  P.  SMITH* 

WILLIAM  T.  GOLDEN  WALTER  S.  VINCENT 

BUILDINGS  AND  GROUNDS  COMMITTEE 

FRANCIS  HOSKIN,  Chairman  CLIFFORD  HARDING,  JR. 

LAWRENCE  B.  COHEN  PHILIP  PERSON 

A.  FARMANFARMAIAN  ROBERT  PRUSCH 

ALAN  FEIN  THOMAS  REESE 

DANIEL  GILBERT  EVELYN  SPIEGEL 

ROBERT  GUNNING*  JAY  WELLS 

COMPUTER  COMMITTEE 

JOHN  HOBBIE,  Chairman  WILLIAM  S.  LITTLE 

WILLIAM  J.  ADELMAN  E.  F.  MACNICHOL,  JR. 

FRANCIS  P.  BOWLES  CONSTANTINE  TOLLIOS 
A.  FARMANFARMAIAN 


4  MARINE  BIOLOGICAL  LABORATORY 

EMPLOYEE  RELATIONS  COMMITTEE 

CATHERINE  NORTON,  Chairman  ROGER  HOBBS 

WILLIAM  EVANS  LEWIS  LAWDAY 

JOHN  HELFRICH  DONALD  LEHY 

HOUSING,  FOOD  SERVICE,  AND  DAY  CARE  COMMITTEE 

ANN  STUART,  Chairman  JOAN  HOWARD 

DANIEL  ALKON  RONALD  JOYNER 

NINA  ALLEN  AIMLEE  LADERMAN 

ROBERT  BARLOW  BRIAN  SALZBERG 

MONA  GROSS  HOMER  P.  SMITH* 

INSTRUCTION  COMMITTEE 

SHELDON  SEGAL,  Chairman  ROBERT  JOSEPHSON 

DANIEL  ALKON  MORTON  MASER* 

ROBERT  ALLEN  MERLE  MIZELL 

JOHN  DOWLING  GEORGE  PAPPAS 

JOHN  HOBBIE  RICHARD  WHITTAKER 
RONALD  HOY 

INVESTMENT  COMMITTEE 

W.  NICHOLAS  THORNDIKE,  Chairman  MAURICE  LAZARUS 

PROSSER  GIFFORD*  ROBERT  MAINER* 

WILLIAM  T.  GOLDEN 

LIBRARY  USERS  COMMITTEE 

EDWARD  ADELBERG,  Chairman  ROBERT  GAGOSIAN 

WILFRED  BRYAN  FREDERICK  GRASSLE 

JOHN  DOWLING  SHINYA  INOUE 

LIBRARY  JOINT  MANAGEMENT  COMMITTEE 

EDWARD  ADELBERG,  Chairman  DEREK  SPENCER 

JOE  KIEBALA  JOHN  STEELE 

MACY  SCHOLARSHIP  COMMITTEE 

WILLIAM  V.  SUTTON,  Chairman  EDGAR  E.  SMITH 

LOWELL  DAVIS  JAMES  TOWNSEL 

MORTON  MASER*  WALTER  S.  VINCENT 

JAMES  PERKINS  CHARLES  WALKER 

MARINE  RESOURCES  COMMITTEE 

SEARS  CROWELL,  Chairman  ROBERT  PRENDERGAST 

CARL  J.  BERG  ROBERT  D.  PRUSCH 

JUNE  HARRIGAN  JOHN  S.  RANKIN 

TOM  HUMPHREYS  JOHN  VALOIS* 

JACK  LEVIN  JONATHAN  WITTENBERG 
CYRUS  LEVINTHAL 


TRUSTEES  AND  STANDING  COMMITTEES 

RADIATION  COMMITTEE 

WALTER  S.  VINCENT,  Chairman  JOHN  HOBBIE 

EUGENE  BELL  ANTHONY  LIUZZI 

FRANCIS  P.  BOWLES  E.  F.  MACNICHOL,  JR. 

RICHARD  L.  CHAPPELL  MORTON  MASER* 

PAUL  DE\VEER  HARRIS  RIPPS 

RESEARCH  SERVICES  COMMITTEE 

NINA  S.  ALLEN,  Chairman  MORTON  MASER* 

JELLE  ATEMA  BRYAN  NOE 

ROBERT  BARLOW  BRUCE  PETERSON 

ROBERT  GOLDMAN  BIRGIT  ROSE 

SAMUEL  S.  KOIDE  SIDNEY  TAMM 

RAYMOND  LASER  JAY  WELLS 

RESEARCH  SPACE  COMMITTEE 

GERALD  FISCHBACH,  Chairman  MORTON  MASER* 

CLAY  ARMSTRONG  JERRY  MELILLO 

JOHN  ARNOLD  ALAN  PEARLMAN 

ARTHUR  DuBois  JOEL  ROSENBAUM 

GEORGE  LANGFORD  JOAN  RUDERMAN 

HANS  LAUFER  BRIAN  SALZBERG 

EDUARDO  MACAGNO  ANN  STUART 

SAFETY  COMMITTEE 

ROBERT  GUNNING,  Chairman*  MORTON  MASER* 

DANIEL  ALKON  RAYMOND  STEPHENS 

LEWIS  LAWDAY  PAUL  STEUDLER 

DONALD  LEHY  FREDERICK  THRASHER 

JANE  LEIGHTON  JAY  WELLS 
E.  F.  MACNICHOL,  JR. 

*  ex  officio 


II.  MEMBERS  OF  THE  CORPORATION 

Including  Action  of  the  1981  Annual  Meeting 
LIFE  MEMBERS 

ABBOTT,  MARIE,  259  High  Street,  R.  D.  2,  Coventry,  CT  06238 

ADOLPH,  EDWARD  F.,  University  of  Rochester,  School  of  Medicine  and  Dentistry,  Rochester, 

NY  14642 

BEAMS,  HAROLD  W.,  University  of  Iowa,  Department  of  Zoology,  Iowa  City,  IA  52242 
BEHRE,  ELLINOR  Black  Mountain,  NC  28711 
BERTHOLF,  LLOYD  M.,  Westminster  Village  #2114,  2025  E.  Lincoln  Street,  Bloomington, 

IL  61701 
BISHOP,  DAVID  W.,  Department  of  Physiology,  Medical  College  of  Ohio,  C.  S.  10008,  Toledo, 

OH  43699 
BOLD,  HAROLD  C.,  Department  of  Botany,  University  of  Texas,  Austin,  TX  78712 


6  MARINE  BIOLOGICAL  LABORATORY 

BRIDGMAN,  A.  JOSEPHINE,  715  Kirk  Road,  Decatur,  GA  30030 

BURBANCK,  MADELINE?.,  Box  15134,  Atlanta,  GA  30333 

BURBANCK,  WILLIAM  D.,  Box  15134,  Atlanta,  GA  30333 

BURDICK,  C.  LALOR,  900  Barley  Drive,  Barley  Mill  Court,  Wilmington,  DE  19807 

CARPENTER,  RUSSELL  L.,  60  Lake  Street,  Winchester,  MA  01890 

CHASE,  AURIN,  Professor  of  Biology,  Emeritus,  Princeton  University,  Princeton,  NJ  08540 

CLARKE,  GEORGE  L.,  44  Juniper  Road,  Belmont,  MA  02178 

CLEMENT,  ANTHONY  C.,  Department  of  Biology,  Emory  University,  Atlanta,  GA  30322 

COLE,  KENNETH  S.,  2404  Loring  Street,  San  Diego,  CA  92109 

COLWIN,  ARTHUR,  320  Woodcrest  Road,  Key  Biscayne,  FL  33149 

COLWIN,  LAURA,  320  Woodcrest  Road,  Key  Biscayne,  FL  33149 

COPELAND,  D.  E.,  41  Fern  Lane,  Woods  Hole,  MA  02543 

COSTELLO,  HELEN  M.,  507  Monroe  Street,  Chapel  Hill,  NC  27514 

CROUSE,  HELEN,  Institute  of  Molecular  Biophysics,  Florida  State  University,  Tallahassee, 

FL  32306 

DILLER,  IRENE  C.,  2417  Fairhill  Avenue,  Glenside,  PA  19038 
DILLER,  WILLIAM  F.,  2417  Fairhill  Avenue,  Glenside,  PA  19038 
ELLIOTT,  ALFRED  M.,  2345  Tarpon  Road,  Naples,  FL  33992 

FERGUSON,  JAMES  K.  W.,  56  Clarkehaven  Street,  Thornhill,  Ontario,  L4J  2B4  Canada 
FRAENKEL,  GOTTFRIED  S.,  Department  of  Entomology,  University  of  Illinois,  320  Morrill 

Hall,  Urbana,  IL  61801 

FRIES,  ERIK,  F.  B.,  3870  Leafy  Way,  Miami,  FL  33133 
OILMAN,  LAUREN  C.,  Department  of  Biology,  University  of  Miami,  PO  Box  24918,  Coral 

Gables,  FL  33124 

GREEN,  JAMES  W.,  Department  of  Physiology,  Rutgers  University,  Piscataway,  NJ  08854 
GRUNDFEST,  HARRY,  Department  of  Neurology,  College  of  Physicians  and  Surgeons,  Co- 
lumbia University,  New  York,  NY  10032 
GUTTMAN,  RITA,  75  Henry  Street,  Brooklyn,  NY  11210 

HAMBURGER,  VIKTOR,  Professor  Emeritus,  Washington  University,  St.  Louis,  MO  63130 
HAMILTON,  HOWARD  L.,  Department  of  Biology,  University  of  Virginia,  Charlottesville, 

VA  22901 

HARTLINE,  H.  KEFFER,  The  Rockefeller  University,  New  York,  NY  10021 
HIBBARD,  HOPE,  143  East  College  Street,  Apt.  309,  Oberlin,  Ohio  44074 
HISAW,  F.  L.,  5925  SW  Plymouth  Drive,  Corvallis,  OR  97330 
HOLLAENDER,  ALEXANDER,  Associated  Universities,  Inc.,  1717  Massachusetts  Avenue,  NW, 

Washington,  DC  20036 

HUMES,  ARTHUR,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
JOHNSON,  FRANK  H.,  Department  of  Biology,  Princeton  University,  Princeton,  NJ  08540 
KAAN,  HELEN,  62  Locust  Street,  Falmouth,  MA  02540 
KAHLER,  ROBERT,  PO  Box  423,  Woods  Hole,  MA  02543 
KILLE,  FRANK  R.,  500  Osceola  Avenue,  Winter  Park,  FL  32789 
KLEINHOLZ,  LEWIS,  Department  of  Biology,  Reed  College,  Portland,  OR  97202 
LOCHHEAD,  JOHN  H.,  49  Woodlawn  Road,  London,  SW6  6PS,  England  U.  K. 
LYNN,  W.  GARDNER,  Department  of  Biology,  Catholic  University  of  America,  Washington, 

DC  20017 

MAGRUDER,  SAMUEL,  R.,  270  Cedar  Lane,  Paducah,  KY  42001 
MANWELL,  REGINALD  D.,  Syracuse  University,  Lyman  Hall,  Syracuse,  NY  13210 
MARSLAND,  DOUGLAS,  Broadmead  N12,  13801  York  Road,  Cockeysville,  MD  21030 
MILLER,  JAMES  A.,  307  Shorewood  Drive,  E.  Falmouth,  MA  02536 
MILNE,  Louis  J.,  Department  of  Zoology,  University  of  New  Hampshire,  Durham,  NH 

03824 

MOORE,  JOHN  A.,  Department  of  Biology,  University  of  California,  Riverside,  CA  92521 
MOUL,  E.  T.,  43  F.  R.  Lillie  Road,  Woods  Hole,  MA  02543 
NACHMANSOHN,  DAVID,  Department  of  Neurology,  College  of  Physicians  and  Surgeons, 

Columbia  University,  630  West  168th  Street,  New  York,  NY  10032 
PAGE,  IRVING  H.,  Box  516,  Hyannisport,  MA  02647 
PLOUGH,  HAROLD  H.,  31  Middle  Street,  Amherst,  MA  01002 


MEMBERS  OF  THE  CORPORATION 

POLLISTER,  A.  W.,  Box  23,  Dixfield,  ME  04224 

POND,  SAMUEL,  E.,  PO  Box  63,  E.  Winthrop,  ME  04343 

PRYTZ,  MARGARET  MCDONALD,  21  Couns  Lane,  Oyster  Bay,  NY  11771 

RANKIN,  JOHN  A.,  JR.,  Box  97,  Ashford,  CT  06278 

RENN,  CHARLES  E.,  Route  2,  Hempstead,  MD  21074 

REZNIKOFF,  PAUL,  1 1  Brooks  Road,  Woods  Hole,  MA  02543 

RICHARDS,  A.  GLENN,  Department  of  Entomology,  Fisheries  and  Wildlife,  University  of 
Minnesota,  St.  Paul,  MN  55101 

RICHARDS,  OSCAR  W.,  Route  1,  Box  79F,  Oakland,  OR  97462 

SCHARRER,  BERTA,  Department  of  Anatomy,  Albert  Einstein  College  of  Medicine,  1300 
Morris  Park  Avenue,  Bronx,  NY  10461 

SCHMITT,  F.  O.,  165  Allendale  Street,  Jamaica  Plain,  MA  02130 

SHEMIN,  DAVID,  Department  of  Biochemistry  and  Molecular  Biology,  Northwestern  Uni- 
versity, Evanston,  IL  60201 

SICHEL,  ELSA,  4  Whitman  Road,  Woods  Hole,  MA  02543 

SONNENBLICK,  B.  P.,  Department  of  Zoology  and  Physiology,  Rutgers  University,  195  Uni- 
versity Avenue,  Newark,  NJ  07102 

SPEIDEL,  CARL,  C,  1873  Field  Road,  Charlottesville,  VA  22903 

STEINHARDT,  JACINTO,  1 508  Spruce  Street,  Berkeley,  CA  94709 

STUNKARD,  HORACE  W.,  American  Museum  of  Natural  History,  Central  Park  West  at  79th 
Street,  New  York,  NY  10024 

TAYLOR,  W.  RANDOLPH,  Department  of  Botany,  University  of  Michigan,  Ann  Arbor,  MI 
48109 

TEWINKEL,  Lois  E.,  4  Sanderson  Avenue,  Northampton,  MA  01060 

TRAVIS,  DOROTHY,  35  Coleridge  Drive,  Falmouth,  MA  02540 

WALD,  GEORGE,  Higgins  Professor  of  Biology,  Emeritus,  Harvard  University,  Cambridge, 
MA  02138 

WICHTERMAN,  RALPH,  31  Buzzards  Bay  Avenue,  Woods  Hole,  MA  02543 

YOUNG,  D.  B.,  Main  Street,  North  Hanover,  NH  02357 

ZINN,  DONALD  J.,  PO  Box  589,  Falmouth,  MA  02541 

ZORZOLI,  ANITA,  Department  of  Botany,  Vassar  College,  Poughkeepsie,  NY  12601 

ZWEIFACH,  BENJAMIN  W.,  %  Ames,  University  of  California,  La  Jolla,  CA  92037 

REGULAR  MEMBERS 

ACHE,  BARRY  W.,  Whitney  Marine  Laboratory,  University  of  Florida,  Rt.  1,  Box  121,  St. 

Augustine,  FL  32084 

ACHESON,  GEORGE  H.,  25  Quissett  Avenue,  Woods  Hole,  MA  02543 
ADEJUWON,  CHRISTOPHER  A.,  Chemical  Pathology  Department,  University  of  Ibadan,  Iba- 

dan,  Nigeria 
ADELBERG,  EDWARD  A.,  Department  of  Human  Genetics,  Yale  University  Medical  School, 

New  Haven,  CT  06511 

AFZELIUS,  BJORN,  Wenner-Gren  Institute,  University  of  Stockholm,  Stockholm,  Sweden 
ALBERTE,  RANDALL  S.,  University  of  Chicago,  Barnes  Laboratory,  5630  S.  Ingleside  Avenue, 

Chicago,  IL  60637 
ALKON,  DANIEL,  Head,  Section  on  Neural  Systems,  Laboratory  of  Biophysics,  NIH,  Marine 

Biological  Laboratory,  Woods  Hole,  MA  02543 

ALLEN,  GARLAND  E.,  Department  of  Biology,  Washington  University,  St.  Louis,  MO  63130 
ALLEN,  NINA  S.,  Department  of  Biology,  Dartmouth  College,  Hanover,  NH  03755 
ALLEN,  ROBERT  D.,  Department  of  Biology,  Dartmouth  College,  Hanover,  NH  03755 
ALSCHER,  RUTH,  Department  of  Biology,  Manhattanville  College,  Purchase,  NY  10577 
AMATNIEK,  ERNEST,  4797  Boston  Post  Road,  Pelham  Manor,  NY  10803 
ANDERSON,  EVERETT,  Department  of  Anatomy,  LHRRB,  Harvard  Medical  School,  45  Shat- 

tuck  Street,  Boston,  MA  02115 

ANDERSON,  J.  M.,  Cornell  University,  Emerson  Hall,  Ithaca,  NY  14850 
ARMSTRONG,  CLAY  M.,  Department  of  Physiology,  University  of  Pennsylvania  Medical 

School,  Philadelphia,  PA  19174 
ARMSTRONG,  PETER  B.,  Department  of  Zoology,  University  of  California,  Davis,  CA  95616 


8  MARINE  BIOLOGICAL  LABORATORY 

ARNOLD,  JOHN  M,  Kewalo  Marine  Laboratory,  University  of  Hawaii,  42  Ahui  Street, 

Honolulu,  HI  96813 

ARNOLD,  WILLIAM  A.,  102  Balsam  Road,  Oak  Ridge,  TN  37830 
ASHTON,  ROBERT  W.,  Gaston  Snow  Beekman  and  Bogue,  14  Wall  Street,  New  York,  NY 

10005 

ATEMA,  JELLE,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
ATWOOD,  KIMBALL  C.,  100  Haven  Avenue,  Apt.  21-E,  New  York,  NY  10032 
AUSTIN,  MARY  L.,  506'/2  North  Indiana  Avenue,  Bloomington,  IN  47401 
BACON,  ROBERT,  PO  Box  723,  Woods  Hole,  MA  02543 

BALDWIN,  THOMAS  O.,  Department  of  Biochemistry  and  Biophysics,  Texas  A  &  M  Uni- 
versity, College  Station,  TX  77843 
BANG,  BETSY,  Johns  Hopkins  University,  School  of  Hygiene  and  Public  Health,  Department 

of  Pathobiology,  Baltimore,  MD  21205 
BARKER,  JEFFERY  L.,  National  Institutes  of  Health,  Bldg.  36  Room  2002,  Bethesda,  MD 

20205 
BARLOW,  ROBERTS.,  JR.,  Institute  for  Sensory  Research,  Syracuse  University,  Merrill  Lane, 

Syracuse,  NY  13210 

BARTELL,  CLELMER  K.,  2000  Lake  Shore  Drive,  New  Orleans,  LA  70122 
BARTH,  LUCENA  J.,  26  Quissett  Avenue,  Woods  Hole,  MA  02543 
BARTLETT,  JAMES  H.,  Department  of  Physics,  Box  1921,  University  of  Alabama,  University, 

AL  35486 

BAUER,  G.  ERIC,  Department  of  Anatomy,  University  of  Minnesota,  Minneapolis,  MN  55414 
BEAUGE,  Luis  ALBERTO,  Institute  de  Investigacion  Medica,  Casilla  de  Correo  389,  5000 

Cordoba,  Argentina 

BECK,  L.  V.,  Department  of  Pharmacology,  School  of  Experimental  Medicine,  Indiana  Uni- 
versity, Bloomington,  IN  47401 

BEGG,  DAVID  A.,  LHRRB,  Harvard  Medical  School,  45  Shattuck  Street,  Boston,  MA  021 15 
BELL,  EUGENE,  Department  of  Biology,  Massachusetts  Institute  of  Technology,  77  Massa- 
chusetts Avenue,  Cambridge,  MA  02139 
BENNETT,  M.  V.  L.,  Albert  Einstein  College  of  Medicine,  Department  of  Neuroscience,  1 300 

Morris  Park  Avenue,  New  York,  NY  10461 

BENNETT,  MIRIAM  F.,  Department  of  Biology,  Colby  College,  Waterville,  ME  04901 
BERG,  CARL  J.,  JR.,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
BERMAN,  MONES,  National  Institutes  of  Health,  Theoretical  Biology  NCI,  Bldg.  10  4B56, 

Bethesda,  MD  20205 

BERNE,  ROBERT  W.,  University  of  Virginia,  School  of  Medicine,  Charlottesville,  VA  22908 
BERNHEIMER,  ALAN  W.,  New  York  University,  School  of  Medicine,  New  York,  NY  10016 
BEZANILLA,  FRANCISCO,  Department  of  Physiology,  University  of  California,  Los  Angeles, 

CA  90052 

BIGGERS,  JOHN  D.,  Department  of  Physiology,  Harvard  Medical  School,  Boston,  MA  021 15 
BISHOP,  STEPHEN  H.,  Department  of  Zoology,  Iowa  State  University,  Ames,  IA  50010 
BLAUSTEIN,  MORDECAI  P.,  Department  of  Physiology,  School  of  Medicine,  University  of 

Maryland,  655  W.  Baltimore  Street,  Baltimore,  MD  21201 
BOETTIGER,  EDWARD  G.,  29  Juniper  Point,  Woods  Hole,  MA  02543 
BOGORAD,  LAWRENCE,  The  Biological  Laboratories,  Harvard  University,  Cambridge,  MA 

02138 
BOOLOOTIAN,  RICHARD  A.,  Science  Software  Systems,  Inc.,  1 1899  West  Pico  Blvd.,  W.  Los 

Angeles,  CA  90064 
BOREI,  HANS  G.,  Department  of  Zoology,  University  of  Pennsylvania,  Philadelphia,  PA 

19174 

BORGESE,  THOMAS,  A.,  Department  of  Biology,  Lehman  College,  CUNY,  Bronx,  NY  10468 
BORISY,  GARY  G.,  Laboratory  of  Molecular  Biology,  University  of  Wisconsin,  Madison,  WI 

53715 

BOSCH,  HERMAN  F.,  moved — no  forwarding  address 

BOTKIN,  DANIEL,  Department  of  Biology,  University  of  California,  Santa  Barbara,  CA  93 106 
BOWEN,  VAUGHN  T.,  Woods  Hole  Oceanographic  Institution,  Redfield  Bldg.  3-32,  Woods 

Hole,  MA  02543 


MEMBERS  OF  THE  CORPORATION 

BOYER,  BARBARA  C,  Department  of  Biology,  Union  College,  Schenectady,  NY  12308 

BOWLES,  FRANCIS  P.,  Ecosystems  Center,  Marine  Biological  Laboratory,  Woods  Hole,  MA 
02543 

BRANDT,  PHILIP  W.,  College  of  Physicians  and  Surgeons,  Department  of  Anatomy,  Columbia 
University,  630  W.  168th  Street,  New  York,  NY  10032 

BRINLEY,  F.  J.,  Neurological  Disorders  Program,  NINCDS,  716  Federal  Building,  Bethesda, 
MD  20205 

BROOKS,  MATILDA  M.,  544  N.  4th  Street,  Corvallis,  OR  97330 

BROWN,  FRANK  A.,  JR.,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

BROWN,  JAY  C.,  Department  of  Neurobiology,  University  of  Virginia,  Charlottesville,  VA 
22908 

BROWN,  JOEL  E.,  Department  of  Physiology  and  Biophysics,  Health  Sciences  Center,  SUNY, 
Stony  Brook,  NY  11794 

BROWN,  STEPHEN  C.,  Department  of  Biological  Sciences,  SUNY,  Albany,  NY  12222 

BUCK,  JOHN  B.,  National  Institutes  of  Health,  Laboratory  of  Physical  Biology,  Bethesda, 
MD  20205 

BURDICK,  CAROLYN  J.,  Department  of  Biology,  Brooklyn  College,  Brooklyn,  NY  11210 

BURGER,  MAX,  Department  of  Biochemistry,  Biocenter  of  the  University  of  Basel,  Klingel- 
bergstrasse  70,  CH-4056,  Basel,  Switzerland 

BURKY,  ALBERT,  Department  of  Biology,  University  of  Dayton,  Dayton,  OH  45469 

BUSH,  LOUISE,  7  Snapper  Lane,  Falmouth,  MA  02540 

CANDELAS,  GRACIELA  C.,  Department  of  Biology,  University  of  Puerto  Rico,  Rio  Piedras, 
PR  00931 

CARLSON,  FRANCIS  D.,  Department  of  Biophysics,  Johns  Hopkins  University,  Baltimore, 
MD  21218 

CASE,  JAMES,  Department  of  Biological  Sciences,  University  of  California,  Santa  Barbara, 
CA93106 

CASSIDY,  REV.  J.  D.,  O.P.,  University  of  Illinois  at  Chicago  Circle,  Department  of  Biological 
Sciences,  Box  4348,  Chicago,  IL  60680 

CEBRA,  JOHN  J.,  Department  of  Biology,  Leidy  Labs,  G-6,  University  of  Pennsylvania,  Phil- 
adelphia, PA  19174 

CHAET,  ALFRED  B.,  University  of  West  Florida,  Pensacola,  FL  32504 

CHAMBERS,  EDWARD  L.,  Department  of  Physiology  and  Biophysics,  University  of  Miami 
School  of  Medicine,  PO  Box  016430,  Miami,  FL  33101 

CHAPPELL,  RICHARD  L.,  Department  of  Biological  Sciences,  Hunter  College,  Box  201,  695 
Park  Avenue,  New  York,  NY  10021 

CHAUNCEY,  HOWARD  H.,  30  Falmouth  Street,  Wellesley  Hills,  MA  02181 

CHENEY,  RALPH  H.,  45  Coleridge  Drive,  Falmouth,  MA  02540 

CHILD,  FRANK  M.,  Department  of  Biology,  Trinity  College,  Hartford,  CT  06106 

CITKOWITZ,  ELENA,  410  Livingston  Street,  New  Haven,  CT  06511 

CLARK,  A.  M.,  Department  of  Biological  Sciences,  University  of  Delaware,  Newark,  DE 
19711 

CLARK,  ELOISE  E.,  National  Science  Foundation,  1800  G  Street,  NW,  Washington,  DC 
20550 

CLARK,  HAYS,  26  Deer  Park  Drive,  Greenwich,  CT  06830 

CLARK,  WALLIS  H.,  JR.,  Aquaculture  Program,  RM  243,  Department  of  Animal  Science, 
University  of  California,  Davis,  CA  95616 

CLAUDE,  PHILIPPA,  Primate  Center,  Capitol  Court,  Madison,  WI  53706 

CLAYTON,  RODERICK  K.,  Cornell  University,  Section  of  Genetics,  Development  and  Phys- 
iology, Ithaca,  NY  14850 

CLOWES,  GEORGE  H.  A.,  JR.,  The  Cancer  Research  Institute,  194  Pilgrim  Road,  Boston, 
MA  02215 

CLUTTER,  MARY,  Cellular  and  Physiological  Biosciences  Section,  National  Science  Foun- 
dation, 1800  G  Street,  Washington,  DC  20550 

COBB,  JEWELL  P.,  President,  California  State  University,  Fullerton,  CA  92634 

COHEN,  ADOLPH  I.,  Department  of  Ophthamology,  School  of  Medicine,  Washington  Uni- 
versity, 660  S.  Euclid  Avenue,  St.  Louis,  MO  63110 


10  MARINE  BIOLOGICAL  LABORATORY 

COHEN,  CAROLYN,  Rosenstiel  Basic  Medical  Sciences  Research  Center,  Brandeis  University, 

Waltham,  MA  02154 
COHEN,  LAWRENCE  B.,  Department  of  Physiology,  Yale  University,  333  Cedar  Street,  New 

Haven,  CT  06510 
COHEN,  SEYMOUR  S.,  Department  of  Pharmacological  Science,  SUNY,  Stony  Brook,  NY 

11790 
COHEN,  WILLIAM  D.,  Department  of  Biological  Sciences,  Hunter  College,  695  Park  Avenue, 

New  York,  NY  10021 

COLLIER,  JACK  R.,  Department  of  Biology,  Brooklyn  College,  Brooklyn,  NY  11210 
COOK,  JOSEPH  A.,  The  Edna  McConnell  Clark  Foundation,  250  Park  Avenue,  New  York, 

NY  10017 
COOPERSTEIN,  S.  J.,  University  of  Connecticut,  School  of  Medicine,  Farmington  Avenue, 

Farmington,  CT  06032 
CORLISS,  JOHN  O.,  Department  of  Zoology,  University  of  Maryland,  College  Park,  MD 

20742 

CORNELL,  NEAL  W.,  6428  Bannockburn  Drive,  Bethesda,  MD  20817 
CORNMAN,  IVOR,  10A  Orchard  Street,  Woods  Hole,  MA  02543 
COSTELLO,  WALTER  J.,  College  of  Medicine,  Ohio  University,  Athens,  OH  45701 
COUCH,  ERNEST  F.,  Department  of  Biology,  Texas  Christian  University,  Fort  Worth,  TX 

76129 

CRANE,  JOHN  O.,  315  West  106th  Street,  New  York,  NY  10025 
CREMER-BARTELS,  GERTRUD,  Universitats  Augenklinik,  44  Munster,  Germany 
CRIPPA,  MARCO,  Department  de  Biologic  animate  Embryologie  Moleculaire,  154  route  de 

Malagnou,  CH  1224  Chene-Bougeries,  Geneve,  Switzerland 
CROW,  TERRY  J.,  Department  of  Physiology,  University  of  Pittsburgh,  School  of  Medicine, 

Pittsburgh,  PA  15261 

CROWELL,  SEARS,  Department  of  Biology,  Indiana  University,  Bloomington,  IN  47401 
DAIGNAULT,  ALEXANDER  T.,  W.  R.  Grace  Company,  1114  Avenue  of  the  Americas,  New 

York,  NY  10036 

DAN,  KATSUMA,  Professor  Emeritus,  Tokyo  Metropolitan  Union,  Meguro-ku,  Tokyo,  Japan 
DANIELLI,  JAMES  F.,  185  Highland  Street,  Worcester,  MA  01609 
DAVIS,  BERNARD  D.,  Bacterial  Physiology  Unit,  Harvard  Medical  School,  25  Shattuck 

Street,  Boston,  MA  02115 

DAW,  NIGEL  W.,  78  Aberdeen  Place,  Clayton,  MO  63105 
DEGROOF,  ROBERT  C,  511  Carpenter  Lane,  Philadelphia,  PA  19119 
DEHAAN,  ROBERT  L.,  Department  of  Anatomy,  Emory  University,  Atlanta,  GA  30322 
DELANNEY,  Louis  E.,  Institute  for  Medical  Research,  751  Bascom  Avenue,  San  Jose,  CA 

95128 

DEPHILLIPS,  HENRY  A.,  JR.,  Department  of  Chemistry,  Trinity  College,  Hartford,  CT  06 1 06 
DETERRA,  NOEL,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

DETTBARN,  WOLF-DIETRICH,  Department  of  Pharmacology,  School  of  Medicine,  Vanderbilt 
University,  Nashville,  TN  37127 

DEWEER,  PAUL  J.,  Department  of  Physiology,  School  of  Medicine,  Washington  University, 

St.  Louis,  MO  63110 
DISCH,  ZACHARIAS,  College  of  Physicians  and  Surgeons,  Columbia  University  Eye  Institute, 

630  W.  165th  Street,  New  York,  NY  10032 
DIXON,  KEITH  E.,  School  of  Biological  Sciences,  Flinders  University,  Bedford  Park,  South 

Australia 
DOWDALL,  MICHAEL  J.,  Department  of  Biochemistry,  University  Hospital  and  Medical 

School,  Nottingham  N672  UH,  England  U.K. 
DOWLING,  JOHN  E.,  The  Biological  Laboratories,  Harvard  University,  16  Divinity  Street, 

Cambridge,  MA  02138 
DRESDEN,  MARC  H.,  Department  of  Biochemistry,  Baylor  College  of  Medicine,  Houston, 

TX  77025 


MEMBERS  OF  THE  CORPORATION  1  1 

DUDLEY,  PATRICIA  L.,  Department  of  Biological  Sciences,  Barnard  College,  Columbia  Uni- 
versity, New  York,  NY  10027 

DUNHAM,  PHILIP  B.,  Department  of  Biology,  Syracuse  University,  Syracuse,  NY  13210 
EBERT,  JAMES  D.,  Office  of  the  President,  Carnegie  Institution  of  Washington,  1 530  P  Street 

NW,  Washington,  DC  20008 
ECKBERG,  WILLIAM,  R.,  Department  of  Zoology,  Howard  University,  Washington,  DC 

20059 
ECKERT,  ROGER  O.,  Department  of  Zoology,  University  of  California,  Los  Angeles,  CA 

90024 
EDDS,  KENNETH  T.,  Department  of  Anatomical  Sciences,  State  University  of  New  York, 

Buffalo,  NY  14214 
EDDS,  LOUISE,  College  of  Osteopathic  Medicine,  Grosvenor  Hall,  Ohio  University,  Athens, 

OH  45701 
EDER,  HOWARD  A.,  Albert  Einstein  College  of  Medicine,  1300  Morris  Park  Avenue,  New 

York,  NY  10461 
EDWARDS,  CHARLES,  Department  of  Biological  Sciences,  State  University  of  New  York, 

Albany,  NY  12222 

EGYUD,  LASZLO  G.,  PO  Box  342,  Woods  Hole,  MA  02543 
EHRENSTEIN,  GERALD,  National  Institutes  of  Health,  Bethesda,  MD  20205 
EHRLICH,  BARBARA  E.,  Department  of  Physiology,  Albert  Einstein  College  of  Medicine, 

1300  Morris  Park  Avenue,  New  York,  NY  10461 

EICHEL,  HERBERT  J.,  226  W.  Rattinghouse  Square,  Philadelphia,  PA  19174 
EISEN,  ARTHUR  Z.,  Chief  of  Division  of  Dermatology,  Washington  University,  St.  Louis, 

MO  63110 

ELDER,  HUGH  YOUNG,  Institute  of  Physiology,  University  of  Glasgow,  Glasgow,  Scotland 
ELLIOTT,  GERALD  F.,  The  Open  University  Research  Unit,  Foxcombe  Hall,  Berkeley  Road, 

Boars  Hill,  Oxford,  England,  U.  K. 

EPEL,  DAVID,  Hopkins  Marine  Station,  Pacific  Grove,  CA  93950 

EPSTEIN,  HERMAN  T.,  Department  of  Biology,  Brandeis  University,  Waltham,  MA  02154 
ERULKAR,  SOLOMON  D.,  318  Kent  Road,  Bala  Cynwyd,  PA  19004 
ESSNER,  EDWARDS.,  Kresge  Eye  Institute,  Wayne  State  University,  540  E.  Canfield  Avenue, 

Detroit,  MI  48201 

ETTIENE,  EARL  M.,  Department  of  Anatomy,  Harvard  Medical  School,  Boston,  MA  02115 
FAILLA,  PATRICIA  M.,  Argonne  National  Laboratory,  Office  of  the  Director,  Argonne,  IL 

60439 
FARMANFARMAIAN,  A.,  Department  of  Physiology  and  Biochemistry,  Rutgers  University, 

New  Brunswick,  NJ  08903 

FAUST,  ROBERT  G.,  Department  of  Physiology,  Medical  School,  University  of  North  Car- 
olina, Chapel  Hill,  NC  27514 
FEIN,  ALAN,  Laboratory  of  Sensory  Physiology,  Marine  Biological  Laboratory,  Woods  Hole, 

MA  02543 
FERGUSON,  F.  P.,  National  Institute  of  General  Medical  Sciences,  National  Institutes  of 

Health,  Bethesda,  MD  20205 

FESSENDEN,  JANE,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
FINKELSTEIN,  ALAN,  Albert  Einstein  College  of  Medicine,  1300  Morris  Park  Avenue,  New 

York,  NY  10461 
FISCHBACH,  GERALD,  Department  of  Anatomy  and  Neurobiology,  Washington  University, 

School  of  Medicine,  St.  Louis,  MO  63110 
FISCHMAN,  DONALD  A.,  Department  of  Anatomy  and  Cell  Biology,  SUNY,  Downstate 

Medical  Center,  450  Clarkson  Avenue,  Brooklyn,  NY  11203 
FISHER,  J.  MANNERY,  Department  of  Biochemistry,  University  of  Toronto,  Toronto,  Ontario, 

Canada  M5S  1A8 
FISHMAN,  HARVEY  M.,  Department  of  Physiology,  University  of  Texas,  Medical  Branch, 

Galveston,  TX  77550 
FLANAGAN,  DENNIS,  Editor,  Scientific  American,  415  Madison  Avenue,  New  York,  NY 

10017 


12  MARINE  BIOLOGICAL  LABORATORY 

Fox,  MAURICE  S.,  Department  of  Biology,  Massachusetts  Institute  of  Technology,  Cam- 
bridge, MA  02139 

FRANZINI,  CLARA,  Department  of  Biology  G-5,  School  of  Medicine,  University  of  Penn- 
sylvania, Philadelphia,  PA  19174 
FRAZIER,  DONALDT.,  Department  of  Physiology  and  Biophysics,  School  of  Medicine,  Temple 

University,  3420  North  Broad  Street,  Philadelphia,  PA  19140 
FREEMAN,  ALAN  R.,  Department  of  Physiology,  Temple  University,  3420  North  Broad 

Street,  Philadelphia,  PA  19140 

FREEMAN,  GARY  L.,  Department  of  Zoology,  University  of  Texas,  Austin,  TX  78172 
FRENCH,  ROBERT  J.,  Department  of  Biophysics,  University  of  Maryland,  School  of  Medicine, 

Baltimore,  MD  21201 

FREYGANG,  WALTER  J.,  JR.,  6247  29th  Street,  NW,  Washington,  DC  20015 
FULTON,  CHANDLER  M.,  Department  of  Biology,  Brandeis  University,  Waltham,  MA  02154 
FURSHPAN,  EDWIN,  J.,  Department  of  Neurophysiology,  Harvard  Medical  School,  Boston, 

MA  02115 
FUSELER,  JOHN  W.,  Department  of  Cell  Biology,  University  of  Texas,  Medical  Branch, 

53233  Harry  Hines  Blvd.,  Dallas,  TX  75235 

FUTRELLE,  ROBERT  P.,  Department  of  Genetics  and  Development,  515  Morrill  Hall,  Uni- 
versity of  Illinois,  505  S.  Goodwin  Avenue,  Urbana,  IL  68101 
FYE,  PAUL,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  MA  02543 
GABRIEL,  MORDECAI,  Department  of  Biology,  Brooklyn  College,  Brooklyn,  NY  11210 
GAINER,  HAROLD,  Head,  Section  of  Functional  Neurochemistry,  National  Institutes  of 

Health,  Bldg.  36,  Room  2A21,  Bethesda,  MD  20205 

GALL,  JOSEPH  G.,  Department  of  Biology,  Yale  University,  New  Haven,  CT  06520 
GELFANT,  SEYMOUR,  Department  of  Dermatology,  Medical  College  of  Georgia,  Augusta, 

GA  30904 

GELPERIN,  ALAN,  Department  of  Biology,  Princeton  University,  Princeton,  NJ  08540 
GERMAN,  JAMES  L.,  III.,  The  New  York  Blood  Center,  310  East  67th  Street,  New  York, 

NY  10021 
GIBBS,  MARTIN,  Institute  for  Photobiology  of  Cells  and  Organelles,  Brandeis  University, 

Waltham,  MA  02154 

GIBSON,  A.  JANE,  Wing  Hall,  Cornell  University,  Ithaca,  NY  14850 
GIFFORD,  PROSSER,  Woodrow  Wilson  International  Center  for  Scholars,  Smithsonian  Build- 
ing, Washington,  DC  20560 
GILBERT,  DANIEL  L.,  National  Institutes  of  Health,  Laboratory  of  Biophysics,  NINCDS, 

Building  36,  Room  2A-29,  Bethesda,  MD  20205 
GIUDICE,  GIOVANNI,  University  of  Palermo,  22  Palermo,  Italy 
GLUSMAN,  MURRAY,  Department  of  Clinical  Psychiatry,  Columbia  University,  722  W.  168th 

Street,  New  York,  NY  10032 

GOLDEN,  WILLIAM  T.,  40  Wall  Street,  New  York,  NY  10005 
GOLDMAN,  DAVID  E.,  63  Loop  Road,  Falmouth,  MA  02540 
GOLDMAN,  ROBERT  D.,  Department  of  Cell  Biology  and  Anatomy,  Northwestern  University, 

303  E.  Chicago  Avenue,  Chicago,  IL  6061 1 
GOLDSMITH,  MARY  H.  M.,  Department  of  Biology,  Kline  Biology  Tower,  Yale  University, 

New  Haven,  CT  06520 
GOLDSMITH,  PAUL,  Laboratory  of  Biochemistry,  NIAMDD,  National  Institutes  of  Health, 

Bethesda,  MD  20205 

GOLDSMITH,  TIMOTHY  H.,  Department  of  Biology,  Yale  University,  New  Haven,  CT  06520 
GOLDSTEIN,  MOISE  H.,  JR.,  Johns  Hopkins  University,  School  of  Medicine,  720  Rutland 

Avenue,  Baltimore,  MD  21205 
GOODMAN,  LESLEY  JEAN,  Department  of  Zoology  and  Comparative  Physiology,  Queen  Mary 

College,  Mile  End  Road,  London,  El  4NS  England,  U.  K. 

GOTTSCHALL,  GERTRUDE  Y.,  315  E.  68th  Street,  Apt.  9-M,  New  York,  NY  10021 
GOUDSMIT,  ESTHER  M.,  Department  of  Biology,  Oakland  University,  Rochester,  MI  48063 
GOULD,  STEPHEN  J.,  Museum  of  Comparative  Zoology,  Harvard  University,  Cambridge, 

MA  02138 
GRAHAM,  HERBERT,  36  Wilson  Road,  Woods  Hole,  MA  02543 


MEMBERS  OF  THE  CORPORATION  1  3 

GRANT,  PHILIP,  Department  of  Biology,  University  of  Oregon,  Eugene,  OR  97403 
GRASS,  ALBERT,  The  Grass  Foundation,  77  Reservoir  Road,  Quincy,  MA  02170 
GRASS,  ELLEN  R.,  The  Grass  Foundation,  77  Reservoir  Road,  Quincy,  MA  02170 
GRASSLE,  JUDITH,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
GREEN,  JONATHAN  P.,  Department  of  Biology,  Roosevelt  University,  430  S.  Michigan  Av- 
enue, Chicago,  IL  60605 
GREENBERG,  MICHAEL  J.,  Department  of  Biological  Sciences,  Florida  State  University, 

Tallahassee,  FL  32306 

GREGG,  JAMES  H.,  Department  of  Zoology,  University  of  Florida,  Gainesville,  FL  3261 1 
GREIF,  ROGER  L.,  Department  of  Physiology,  Cornell  University,  Medical  College,  New 

York,  NY  10021 

GRIFFIN,  DONALD  R.,  The  Rockefeller  University,  1230  York  Avenue,  New  York,  NY  10021 
GROSCH,  DANIEL  S.,  Department  of  Genetics,  Gardner  Hall,  North  Carolina  State  Uni- 
versity, Raleigh,  NC  27607 
GROSS,  PAUL  R.,  President  and  Director,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 

GROSSMAN,  ALBERT,  New  York  University  Medical  School,  New  York,  NY  10016 
GUNNING,  A.  ROBERT,  377  Hatchville  Road,  Hatchville,  MA  02536 
GWILLIAM,  G.  P.,  Department  of  Biology,  Reed  College,  Portland,  OR  97202 
HALL,  ZACK  W.,  Department  of  Physiology,  University  of  California,  San  Francisco,  CA 

94143 
HALVORSON,  HARLYN  O.,  Rosenstiel  Basic  Medical  Sciences  Research  Center,  Brandeis 

University,  Waltham,  MA  02154 
HAMKALO,  BARBARA  A.,  Department  of  Molecular  Biology  and  Biochemistry,  University 

of  California,  Irvine,  CA  92717 
HANNA,  ROBERTS.,  SUNY,  College  of  Environmental  Science  and  Forestry,  Syracuse,  NY 

13210 
HARDING,  CLIFFORD  V.  JR.,  Kresege  Eye  Institute,  Wayne  State  University,  540  E.  Canfield, 

Detroit,  MI  48210 
HAROSI,  FERENC  I.,   Laboratory  of  Sensory  Physiology,  Marine  Biological  Laboratory, 

Woods  Hole,  MA  02543 
HARRIGAN,  JUNE  F.,  Laboratory  of  Biophysics,  Marine  Biological  Laboratory,  Woods  Hole, 

MA  02543 
HARRINGTON,  GLENN  W.,  Department  of  Microbiology,  School  of  Dentistry,  University  of 

Missouri,  650  E.  25th  Street,  Kansas  City,  MO  64108 
HASCHEMEYER,  AUDREY  E.  V.,  Department  of  Biological  Sciences,  Hunter  College,  695 

Park  Avenue,  New  York,  NY  10021 

HASTINGS,  J.  W.,  The  Biological  Laboratories,  Harvard  University,  Cambridge,  MA  02138 
HAYES,  RAYMOND  L.,  JR.,  Department  of  Anatomy,  School  of  Medicine,  Morehouse  College, 

223  Chestnut  St.,  NW,  Atlanta,  GA  30314 
HAYASHI,  TERU,  7105  SW  112  Place,  Miami,  FL  33173 

HENLEY,  CATHERINE,  7401  Westlake  Terrace,  Apt.  No.  1516,  Bethesda,  MD  20034 
HERNDON,  WALTER  R.,  University  of  Tennessee,  506  Andy  Holt  Tower,  Knoxville,  TN 

37916 

HERVEY,  JOHN  P.,  Box  85,  Penzance  Point,  Woods  Hole,  MA  02543 
HESSLER,  ANITA  Y.,  5795  Waverly  Avenue,  La  Jolla,  CA  92037 
HEUSER,  JOHN,  Department  of  Biophysics,  Washington  University  School  of  Medicine,  St. 

Louis,  MO  63110 
HIATT,  HOWARD  H.,  Office  of  the  Dean,  Harvard  School  of  Public  Health,  677  Huntington 

Avenue,  Boston,  MA  02115 
HIGHSTEIN,  STEPHEN  M.,  Division  of  Cellular  Neurobiology,  Albert  Einstein  College  of 

Medicine,  1300  Morris  Park  Avenue,  Bronx,  NY  10461 
HILDEBRAND,  JOHN  G.,  Department  of  Biological  Sciences,  Fairchild  Center  #9 1 3,  Columbia 

University,  New  York,  NY  10027 

HILL,  ROBERT  B.,  Department  of  Zoology,  University  of  Rhode  Island,  Kingston,  RI  02881 
HILLMAN,  PETER,  Department  of  Biology,  Hebrew  University,  Jerusalem,  Israel 


14  MARINE  BIOLOGICAL  LABORATORY 

HINEGARDNER,  RALPH  T.,  Division  of  Natural  Sciences,  University  of  California,  Santa 

Cruz,  CA  95064 
HINSCH,  GERTRUDE  W.,  Department  of  Biology,  University  of  South  Florida,  Tampa,  FL 

33620 
HOBBIE,  JOHN  E.,  Ecosystems  Center,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 

HODGE,  ALAN  J.,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
HODGE,  CHARLES,  IV,  PO  Box  4095,  Philadelphia,  PA  19118 
HOFFMAN,  JOSEPH,  Department  of  Physiology,  School  of  Medicine,  Yale  University,  New 

Haven,  CT06515 

HOLLYFIELD,  JOE  G.,  Baylor  School  of  Medicine,  Texas  Medical  Center,  Houston,  TX  77030 
HOLTZMAN,  ERIC,  Department  of  Biological  Sciences,  Columbia  University,  New  York,  NY 

10027 

HOLZ,  GEORGE  G.,  JR.,  Department  of  Microbiology,  SUNY,  Syracuse,  NY  13210 
HOSKIN,  FRANCIS  C.  G.,  Department  of  Biology,  Illinois  Institute  of  Technology,  Chicago, 

IL  60616 
HOUGHTON,  RICHARD  A.,  Ill,  Ecosystems  Center,  Marine  Biological  Laboratory,  Woods 

Hole,  MA  02543 

HOUSTON,  HOWARD,  Preston  Avenue,  Meridan,  CT  06450 
HOY,  RONALD  R.,  Section  of  Neurobiology  and  Behavior,  Cornell  University,  Ithaca,  NY 

14850 

HUBBARD,  RUTH,  The  Biological  Laboratories,  Harvard  University,  Cambridge,  MA  02138 
HUFNAGEL,  LINDA  A.,  Department  of  Microbiology,  University  of  Rhode  Island,  Kingston, 

RI  02881 

HUMMON,  WILLIAM  D.,  Department  of  Zoology,  Ohio  University,  Athens,  Ohio  45701 
HUMPHREYS,  SUSIE  H.,  Gerontology  Research  Center,  NIA,  NIH,  Baltimore  City  Hospital, 

Baltimore,  MD  21224 
HUMPHREYS,  TOM  D.,  University  of  Hawaii,  PBRC,  41  Ahui  Street,  Honolulu,  Hawaii 

96813 

HUNTER,  BRUCE  W.,  Box  321,  Lincoln  Center,  MA  01773 
HUNTER,  ROBERT  D.,  Department  of  Biological  Sciences,  Oakland  University,  Rochester, 

NY  48063 

HUNZIKER,  HERBERT  E.,  Esq.,  PO  Box  547,  Falmouth,  MA  02541 

HURWITZ,  CHARLES,  Basic  Science  Research  Lab,  Veterans  Administration  Hospital,  Al- 
bany, NY  12208 
HURWITZ,  JERARD,  Albert  Einstein  College  of  Medicine,  Department  of  Molecular  Biology, 

1300  Morris  Park  Avenue,  Bronx,  NY  10461 

HUXLEY,  HUGH  E.,  Medical  Research  Council,  Laboratory  of  Molecular  Biology,  Cam- 
bridge, England,  U.  K. 
HYNES,  THOMAS  J.,  JR.,  Office  of  the  Senior  Vice  President,  Meredith  and  Grew,  Inc.,  125 

High  Street,  Boston,  MA  02110 
ILAN,  JOSEPH,  Department  of  Anatomy,  Case  Western  Reserve  University,  Cleveland,  OH 

44106 
INOUE,  SADUYKI,  Electron  Microscopy  Laboratory,  McGill  University  Cancer  Center,  655 

Drummond  Street,  Montreal,  P.  A.,  Canada  HG3  1Y6 
INOUE,  SHINYA,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
ISENBERG,  IRVING,  Department  of  Biochemistry  and  Biophysics,  Oregon  State  University, 

Corvallis,  OR  97331 

ISSELBACHER,  KURT  J.,  Massachusetts  General  Hospital,  Boston,  MA  02114 
ISSADORIDES,  MARIETTA  R.,  Department  of  Psychiatry,  University  of  Athens,  Monis  Petraki 

8,  Athens  140,  Greece 

IZZARD,  COLIN  S.,  Department  of  Biological  Sciences,  SUNY,  Albany,  NY  12222 
JACOBSON,  ANTONE  G.,  Department  of  Zoology,  University  of  Texas,  Austin,  TX  78712 
JAFFEE,  LIONEL,  Department  of  Biology,  Purdue  University,  Lafayette,  IN  47907 
JAHAN-PARWAR,  BEHRUS,  Worcester  Foundation  for  Experimental  Biology,  222  Maple 

Avenue,  Shrewsbury,  MA  01545 


MEMBERS  OF  THE  CORPORATION  1  5 

JANNASCH,  HOLGER  W.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  MA  02543 
JEFFERY,  WILLIAM  R.,  Department  of  Zoology,  University  of  Texas,  Austin,  TX  78712 
JENNER,  CHARLES  E.,  Department  of  Zoology,  University  of  North  Carolina,  Chapel  Hill, 

NC  27514 
JENNINGS,  JOSEPH  B.,  Department  of  Zoology,  Baines  Wing,  University  of  Leeds,  Leeds 

LS2  9-JT,  England,  U.  K. 

JONES,  MEREDITH  L.,  Smithsonian  Institution,  Division  of  Worms,  Washington,  DC  20650 
JONES,  RAYMOND  F.,  Department  of  Biology,  SUNY,  Stony  Brook,  NY  11790 
JOSEPHSON,  ROBERT  K.,  School  of  Biological  Sciences,  University  of  California,  Irvine,  CA 

92664 

JOYNER,  RONALD  W.,  Department  of  Physiology,  University  of  Iowa,  Iowa  City,  IA  52242 
KABAT,  E.  A.,  Department  of  Microbiology,  College  of  Physicians  and  Surgeons,  Columbia 

University,  630  West  168th  Street,  New  York,  NY  10032 
KAFATOS,  FOTIS  C,  The  Biological  Laboratories,  Harvard  University,  Cambridge,  MA 

02138 
KALEY,  GABOR,  Department  of  Physiology,  Basic  Sciences  Bldg.,  New  York  Medical  College, 

Valhalla,  NY  10595 
KALTENBACH,  JANE,  Department  of  Biological  Sciences,  Mount  Holyoke  College,  South 

Hadley,  MA  01075 
KAMINER,  BENJAMIN,  Department  of  Physiology,  School  of  Medicine,  Boston  University, 

80  East  Concord  Street,  Boston,  MA  02118 

KAMMER,  ANN  E.,  Division  of  Biology,  Kansas  State  University,  Manhatten,  KS  66506 
KANE,  ROBERT  E.,  University  of  Hawaii,  PBRC,  41  Ahui  Street,  Honolulu,  Hawaii  96813 
KANESHIRO,  EDNA  S.,  Department  of  Biological  Sciences,  University  of  Cincinnati,  Cincin- 
nati, OH  45221 

KAPLAN,  EHUD,  The  Rockefeller  University,  1230  York  Avenue,  New  York,  NY  10021 
KARAKASHIAN,  STEPHEN  J.,  165  West  91st  Street,  Apt.  16-F,  New  York,  NY  10024 
KARUSH,  FRED,  Department  of  Microbiology,  School  of  Medicine,  University  of  Pennsyl- 
vania, Philadelphia,  PA  19174 
KATZ,  GEORGE  M.,  Department  of  Neurology,  College  of  Physicians  and  Surgeons,  Columbia 

University,  630  West  168th  Street,  New  York,  NY  10032 
KEAN,  EDWARD  L.,  Case  Western  Reserve  University,  Department  of  Ophthalmology  and 

Biochemistry,  Cleveland,  Ohio  44101 
KELLY,  ROBERT  E.,  Department  of  Anatomy,  College  of  Medicine,  University  of  Illinois, 

PO  Box  6998,  Chicago,  IL  60680 

KEMP,  NORMAN  E.,  Department  of  Zoology,  University  of  Michigan,  Ann  Arbor,  MI  48104 
KENDALL,  JOHN  P.,  Fanueil  Hall  Associates,  One  Boston  Place,  Boston,  MA  02108 
KETCHUM,  BOSTWICK  H.,  PO  Box  32,  Woods  Hole,  MA  02543 
KEYNAN,  ALEXANDER,  Vice  President,  Hebrew  University,  Jerusalem,  Israel 
KING,  THOMAS  J.,  Division  of  Cancer  Research  Resources  and  Center,  National  Institutes 

of  Health,  Bldg.  31,  Room  10A03,  Bethesda,  MD  20205 

KINGSBURY,  JOHN  M.,  Department  of  Botany,  Cornell  University,  Ithaca,  NY  14853 
KIRSCHENBAUM,  DONALD,  Department  of  Biochemistry,  SUNY,  450  Clarkson  Avenue, 

Brooklyn,  NY  11203 

KLEIN,  MORTON,  Department  of  Microbiology,  Temple  University,  Philadelphia,  PA  19122 
KLOTZ,  I.  M.,  Department  of  Chemistry,  Northwestern  University,  Evanston,  IL  60201 
KOIDE,  SAMUEL  S.,  Population  Council,  The  Rockefeller  University,  66th  Street  and  York 

Avenue,  New  York,  NY  10021 
KONINGSBERG,  IRWIN  R.,  Department  of  Biology,  Gilmer  Hall,  University  of  Virginia, 

Charlottesville,  VA  22903 

KOSOWER,  EDWARD  M.,  Department  of  Chemistry,  Tel  Aviv  University,  Tel  Aviv,  Israel 
KRAHL,  M.  E.,  2783  W.  Casas  Circle,  Tucson,  AZ  85741 
KRANE,  STEPHEN  M.,  Massachusetts  General  Hospital,  Boston,  MA  02114 
KRASSNER,  STUART  M.,  Department  of  Developmental  and  Cell  Biology,  University  of 

California,  Irvine,  CA  92717 
KRAUSS,  ROBERT,  FASEB,  9650  Rockville  Pike,  Bethesda,  MD  20205 


16  MARINE  BIOLOGICAL  LABORATORY 

KRAVITZ,  EDWARD  A.,  Department  of  Neurobiology,  Harvard  Medical  School,  25  Shattuck 

Street,  Boston,  MA  02115 
KRIEBEL,  MAHLON  E.,  Department  of  Physiology,  B.S.B.,  Upstate  Medical  Center,  766 

Irving  Avenue,  Syracuse,  NY  13210 

KRIEG,  WENDELL  J.  S.,  1236  Hinman,  Evanston,  IL  60602 
KUHNS,  WILLIAM  J.,  University  of  North  Carolina,  512  Faculty  Lab  Office  Bldg.,  231-H, 

Chapel  Hill,  NC  27514 
KUSANO,  KIYOSHI,  Illinois  Institute  of  Technology,  Department  of  Biology,  3300  South 

Federal  Street,  Chicago,  I L  60616 

LAMARCHE,  PAUL  H.,  Eastern  Maine  Medical  Center,  489  State  Street,  Bangor,  ME  04401 
LANDIS,  DENNIS  M.  D.,  Department  of  Neurology,  Massachusetts  General  Hospital,  Boston, 

MA  02114 
LANDOWNE,  DAVID,  Department  of  Physiology,  University  of  Miami,  R-430,  PO  Box 

016430,  Miami,  FL  33101 
LANGFORD,  GEORGE  M.,  Department  of  Physiology,  University  of  North  Carolina,  Medical 

Sciences  Research  Wing  206H,  Chapel  Hill,  NC  27514 
LASH,  JAMES  W.,  Department  of  Anatomy,  School  of  Medicine,  University  of  Pennsylvania, 

Philadelphia,  PA  19174 
LASTER,  LEONARD,  President,  University  of  Oregon,  Health  Sciences  Center,  Portland,  OR 

97201 
LAUFER,  HANS,  Biological  Sciences  Group  U-42,  University  of  Connecticut,  Storrs,  CT 

06268 
LAUFFER,  MAX  A.,  Department  of  Biophysics,  University  of  Pittsburgh,  Pittsburgh,  PA 

15260 
LAWRENCE,  E.  SWIFT,  Pawtucket  Institute  for  Savings,  296  Main  Street,  Pawtucket,  RI 

02860 

LAZAROW,  JANE,  221  Woodlawn  Avenue,  St.  Paul,  MN  55106 

LAZARUS,  MAURICE,  Federated  Department  Stores,  Inc.,  50  Cornhill,  Boston,  MA  02108 
LEADBETTER,  EDWARD  R.,  Biological  Sciences  Group  U-42,  University  of  Connecticut, 

Storrs,  CT  06268 

LEAK,  LEE  VIRN,  Department  of  Anatomy,  Howard  University,  Washington,  DC  20001 
LECAR,  HAROLD,  Laboratory  of  Biophysics,  NINCDS,  National  Institutes  of  Health,  Be- 

thesda,  MD  20205 

LEDERBERG,  JOSHUA,  President,  The  Rockefeller  University,  New  York,  NY  10021 
LEDERHENDLER,  IZJA  I.,  Laboratory  of  Biophysics,  Marine  Biological  Laboratory,  Woods 

Hole,  MA  02543 
LEE,  JOHN  J.,  Department  of  Biology,  City  College,  Convent  Avenue  and  138th  Street,  New 

York,  NY  10031 
LEFEVRE,  PAUL  G.,  Department  of  Physiology,  Health  Sciences  Center,  East  Campus— 

SUNY,  Stony  Brook,  NY  1 1794 
LEIGHTON,  JOSEPH,  Department  of  Pathology,  Medical  College  of  Pennsylvania,  3300  Henry 

Avenue,  Philadelphia,  PA  19129 

LEIGHTON,  STEPHEN,  National  Institutes  of  Health,  Bldg.  13  3W13,  Bethesda,  MD  20205 
LENHER,  SAMUEL,  50-C  Cokesbury  Village,  Hockessin,  DE  19707 
LERMAN,  SIDNEY,  Laboratory  for  Ophthalmic  Research,  Emory  University,  Atlanta,  GA 

30322 

LERNER,  AARON  B.,  Yale  Medical  School,  New  Haven,  CT  06510 
LEVIN,  JACK,  Clinical  Pathology  Service,  Veterans  Administration  Hospital — 113A,  4150 

Clement  Street,  San  Francisco,  CA  94120 
LEVINE,  RACHMIEL,  2024  Canyon  Road,  Arcadia,  CA  91006 
LEVINTHAL,  CYRUS,  Department  of  Biological  Sciences,  Columbia  University,  908  Scher- 

merhorn  Hall,  New  York,  NY  10027 
LEVITAN,  HERBERT,  Department  of  Zoology,  University  of  Maryland,  College  Park,  MD 

20742 
LING,  GILBERT,  307  Berkeley  Road,  Marion,  PA  19066 


MEMBERS  OF  THE  CORPORATION  1  7 

LIPICKY,  RAYMOND  J.,  Laboratory  of  Biophysics,  National  Institutes  of  Health,  Bldg.  36, 
Room  2A29,  Bethesda,  MD  20205 

LITTLE,  E.  P.,  216  Highland  Street,  West  Newton,  MA  02158 

Liuzzi,  ANTHONY,  Department  of  Physics,  University  of  Lowell,  Lowell,  MA  01854 

LLINAS,  RODOLFO  R.,  Department  of  Physiology  and  Biophysics,  New  York  University 
Medical  Center,  550  First  Avenue,  New  York,  NY  10016 

LOEWENSTEIN,  WERNER  R.,  Department  of  Physiology  and  Biophysics,  University  of  Miami, 
PO  Box  016430,  Miami,  FL  33101 

LOEWUS,  FRANK  A.,  Department  of  Agricultural  Chemistry,  Washington  State  University, 
Pullman,  WA  99164 

LOFTFIELD,  ROBERT  B.,  Department  of  Biochemistry,  School  of  Medicine,  University  of 
New  Mexico,  900  Stanford,  NE,  Albuquerque,  NM  87105 

LONDON,  IRVING  M.,  Massachusetts  Institute  of  Technology,  1 6-5 1 2,  Cambridge,  MA  02 1 38 

LONGO,  FRANK  J.,  Department  of  Anatomy,  University  of  Iowa,  Iowa  City,  IA  52442 

LORAND,  LASZLO,  Department  of  Biochemistry  and  Molecular  Biology,  Northwestern  Uni- 
versity, Evanston,  IL  60201 

LURIA,  SALVADOR  E.,  Department  of  Biology,  Massachusetts  Institute  of  Technology,  Cam- 
bridge, MA  02139 

LYNCH,  CLARA  J.,  4800  Fillmore  Avenue,  Alexandria,  VA  22311 

MACAGNO,  EDUARDO  R.,  1003B  Fairchild,  Columbia  University,  New  York,  NY  10022 

MAcNiCHOL,  E.  F.,  JR.,  Laboratory  of  Sensory  Physiology,  Marine  Biological  Laboratory, 
Woods  Hole,  MA  02543 

MAHLER,  ROBERT,  Department  of  Biochemistry,  Indiana  University,  Bloomington,  IN  47401 

MAINER,  ROBERT,  Senior  Vice  President,  The  Boston  Company,  One  Boston  Place,  Boston, 
MA  02108 

MALKIEL,  SAUL,  Sidney  Farber  Cancer  Center,  35  Binney  Street,  Boston,  MA  02116 

MANALIS,  RICHARD  S.,  RR  #4,  Columbia  City,  IN  46725 

MANGUM,  CHARLOTTE  P.,  Department  of  Biology,  College  of  William  and  Mary,  Williams- 
burg,  VA  23185 

MARSH,  JULIAN  B.,  Department  of  Biochemistry  and  Physiology,  Medical  College  of  Penn- 
sylvania, 3300  Henry  Avenue,  Philadelphia,  PA  19129 

MARTIN,  LOWELL  V.,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

MARUO,  TAKESHI,  Department  of  Obstetrics  and  Gynecology,  Kobe  University  Ikuta-ku, 
Kobe  650,  Japan 

MASER,  MORTON,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

MASTROIANNI,  LUIGI,  JR.,  Department  of  Obstetrics  and  Gynecology,  University  of  Penn- 
sylvania, Philadelphia,  PA  19174 

MATHEWS,  RITA  W.,  Hunter  College,  Box  1075,  695  Park  Avenue,  New  York,  NY  10021 

MAUTNER,  HENRY  G.,  Department  of  Biochemistry  and  Pharmacology,  Tufts  University, 
136  Harrison  Avenue,  Boston,  MA  02111 

MAUZERALL,  DAVID,  The  Rockefeller  University,  66th  Street  and  York  Avenue,  New  York, 
NY  10021 

MAXWELL,  ARTHUR,  Institute  for  Geophysics,  University  of  Texas,  Austin,  TX  78712 

MAZIA,  DANIEL,  Hopkins  Marine  Station,  Pacific  Grove,  CA  93950 

McCANN,  FRANCES,  Department  of  Physiology,  Dartmouth  Medical  School,  Hanover,  NH 
03755 

McCLOSKEY,  LAWRENCE  R.,  Department  of  Biology,  Walla  Walla  College,  College  Place, 
WA  99324 

MCLAUGHLIN,  JANE  A.,  PO  Box  187,  Woods  Hole,  MA  02543 

McMAHON,  ROBERT  F.,  Department  of  Biology,  Box  19498,  University  of  Texas,  Arlington, 
TX  76019 

MCREYNOLDS,  JOHN  S.,  Department  of  Physiology,  University  of  Michigan,  Ann  Arbor, 
MI  48104 

MEEDEL,  THOMAS,  Boston  University  Marine  Program,  Marine  Biological  Laboratory, 
Woods  Hole,  MA  02543 


18  MARINE  BIOLOGICAL  LABORATORY 

MEINERTZHAGEN,  IAN  A.,  Department  of  Psychology,  Life  Sciences  Center,  Dalhousie  Uni- 
versity, Halifax,  Nova  Scotia,  Canada  B3H  451 
MEINKOTH,  NORMAN  A.,  Department  of  Biology,  Swarthmore  College,  Swarthmore,  PA 

19081 

MEISS,  DENNIS  E.,  Department  of  Biology,  Clark  University,  Worcester,  MA  01610 
MELILLO,  JERRY  M.,  Ecosystems  Center,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 
MELLON,  DEFOREST,  JR.,  Department  of  Biology,  University  of  Virginia,  Charlottesville, 

VA  22903 

MELLON,  RICHARD  P.,  PO  Box  187,  Laughlintown,  PA  15655 
METUZALS,  JANIS,  Department  of  Anatomy,  Faculty  of  Medicine,  University  of  Ottawa, 

Ottawa,  Ontario,  Canada  KIN  9A9 
METZ,  CHARLES  B.,  Institute  for  Molecular  and  Cellular  Evolution,  University  of  Miami, 

521  Anastasia  Avenue,  Coral  Gables,  FL  33134 
MIDDLEBROOK,  ROBERT,  86  Station  Road,  Burley-in-Warfedale,  West  Yorks,  England, 

U.  K. 

MILKMAN,  ROGER,  Department  of  Zoology,  University  of  Iowa,  Iowa  City,  IA  52242 
MILLS,  ERIC  L.,  Institute  of  Oceanography,  Dalhousie  University,  Halifax,  Nova  Scotia 
MILLS,  ROBERT,  56  Worcester  Court,  Falmouth,  MA  02540 
MITCHELL,  RALPH,  Pierce  Hall,  Harvard  University,  Cambridge,  MA  02138 
MIZELL,  MERLE,  Department  of  Biology,  Tulane  University,  New  Orleans,  LA  70118 
MONROY,  ALBERTO,  Stazione  Zoologica,  Villa  Communale,  Napoli,  Italy 
MONTROLL,  ELIOTT  W.,  Institute  for  Fundamental  Studies,  Department  of  Physics,  Roch- 
ester, NY  14627 
MOORE,  JOHN  W.,  Department  of  Physiology,  Duke  University,  Medical  Center,  Durham, 

NC  27710 
MOORE,  LEE  E.,  Department  of  Physiology  and  Biophysics,  University  of  Texas,  Medical 

Branch,  Galveston,  TX  77550 

MORAN,  JOSEPH  F.,  JR.,  23  Foxwood  Drive,  RR#1,  Eastham,  MA  02642 
MORIN,  JAMES  G.,  Department  of  Biology,  University  of  California,  Los  Angeles,  CA  90024 
MORRELL,  FRANK,  Department  of  Neurological  Sciences,  Rush  Medical  Center,  1753  W. 

Congress  Parkway,  Chicago,  IL  60612 

MORRILL,  JOHN  B.,  JR.,  Division  of  Natural  Sciences,  New  College,  Sarasota,  FL  33580 
MORSE,  RICHARD  S.,  193  Winding  River  Road,  Wellesley,  MA  02181 
MORSE,  ROBERT  W.,  Associate  Director,  Woods  Hole  Oceanographic  Institution,  Woods 

Hole,  MA  02543 
MOSCONA,  A.  A.,  Department  of  Biology,  University  of  Chicago,  920  East  58th  Street, 

Chicago,  IL  60637 

MOTE,  MICHAEL  I.,  Department  of  Biology,  Temple  University,  Philadelphia,  PA  19122 
MOUNTAIN,  ISABEL  Vinson  Hall  #112,  6251  Old  Dominion  Drive,  McLean,  VA  22101 
MULLEN,  GEORGE,  President,  Mohawk  Carpets,  Amsterdam,  NY  12010 
MUSACCHIA,  XAVIER  J.,  Graduate  School,  University  of  Louisville,  Louisville,  KY  40295 
NABRIT,  S.  M.,  686  Beckwith  Street,  SW,  Atlanta,  GA  30314 
NACE,  PAUL  F.,  5  Bowditch  Road,  Woods  Hole,  MA  02543 

NAKAJIMA,  SHIGEHIRO,  Department  of  Biological  Sciences,  Purdue  University,  West  La- 
fayette, IN  47907 
NAKAJIMA,  YASUKO,  Department  of  Biological  Sciences,  Purdue  University,  West  Lafayette, 

IN  47907 

NARAHASHI,  TOSHIO,  Department  of  Pharmacology,  Medical  Center,  Northwestern  Uni- 
versity, 303  East  Chicago  Avenue,  Chicago,  IL  60611 

NASATIR,  MAIMON,  Department  of  Biology,  University  of  Toledo,  Toledo,  OH  43606 
NELSON,  LEONARD,  Medical  College  of  Ohio,  Department  of  Physiology,  Toledo,  OH  43699 
NELSON,  MARGARET  C.,  Section  on  Neurobiology  and  Behavior,  Cornell  University,  Ithaca, 

NY  14850 

NICHOLLS,  JOHN  G.,  Department  of  Neurobiology,  Stanford  University,  Stanford,  CA  94305 
NICOSIA,  SANTO  V.,  Department  of  OB-GYN,  Division  of  Reproductive  Biology,  University 
of  Pennsylvania,  Philadelphia,  PA  19174 


MEMBERS  OF  THE  CORPORATION  1  9 

NIELSEN,  JENNIFER  B.  K.,  Waksman  Institute  for  Microbiology,  Piscataway,  NJ  08854 
NOE,  BRYAN  D.,  Department  of  Anatomy,  Emory  University,  Atlanta,  GA  30345 
OBAID,  ANA  LIA,  Department  of  Physiology  and  Pharmacy,  University  of  Pennsylvania, 

School  of  Dental  Medicine,  4001  Spruce  Street,  Philadelphia,  PA  19104 
OCHOA,  SEVERO,  530  East  72nd  Street,  New  York,  NY  10021 
ODUM,  EUGENE,  Department  of  Zoology,  University  of  Georgia,  Athens,  GA  30701 
OERTEL,  DONATA,  Department  of  Neurophysiology,  University  of  Wisconsin,  283  Medical 

Science  Building,  Madison,  WI  53706 
O'HERRON,  JONATHAN,  Lazard  Freres  and  Company,  1  Rockefeller  Plaza,  New  York,  NY 

10020 

O'MELIA,  ANNE  F.,  George  Mason  University,  4400  University  Drive,  Fairfax,  VA  22030 
OLSON,  JOHN  M.,  Department  of  Biology,  Brookhaven  National  Laboratory,  Upton,  NY 

11973 

OSCHMAN,  JAMES  L.,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
PALMER,  DOUGLAS  W.,  21  Stanford  Road,  Wellesley,  MA  02181 
PALMER,  JOHN  D.,  Department  of  Zoology,  University  of  Massachusetts,  Amherst,  MA 

01002 
PALTI,  YORAM,  Department  of  Physiology  and  Biophysics,  Israel  Institute  of  Technology, 

12  Haaliya  Street,  BAT-GALIM,  POB  9649,  Haifa,  Israel 
PAPPAS,  GEORGE  D.,  Department  of  Anatomy,  College  of  Medicine,  University  of  Illinois, 

808  South  Wood  Street,  Chicago,  IL  60612 
PARDEE,  ARTHUR  B.,  Department  of  Pharmacology,  Harvard  Medical  School,  Boston,  MA 

02115 

PARDY,  ROSEVELT  L.,  School  of  Life  Sciences,  University  of  Nebraska,  Lincoln,  NE  27710 
PARMENTIER,  JAMES  L.,  Department  of  Anesthesiology,  Duke  University  Medical  Center, 

Durham,  NC  27710 
PASSANO,  LEONARD  M.,  Department  of  Zoology,  Birge  Hall,  University  of  Wisconsin, 

Madison,  WI  53706 

PEARLMAN,  ALAN  L.,  Department  of  Physiology,  School  of  Medicine,  Washington  Univer- 
sity, St.  Louis,  MO  63110 
PEDERSON,  THORU,  Worcester  Foundation  for  Experimental  Biology,  Shrewsbury,  MA 

01545 

PERKINS,  C.  D.,  National  Academy  of  Engineering,  2101  Constitution  Avenue,  NW,  Wash- 
ington, DC  20418 

PERSON,  PHILIP,  Chief,  Special  Dental  Research  Program,  Veterans  Administration  Hos- 
pital, Brooklyn,  NY  11219 
PETERSON,  BRUCE  J.,  Ecosystems  Center,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 
PETTIBONE,  MARIAN  H.,  Division  of  Worms,  W-213,  Smithsonian  Institution,  Washington, 

DC  20560 

PFOHL,  RONALD  J.,  Department  of  Zoology,  Miami  University,  Oxford,  OH  45056 
PIERCE,  SIDNEY  K.,  JR.,  Department  of  Zoology,  University  of  Maryland,  College  Park,  MD 

20740 
POLLARD,  HARVEY  B.,  National  Institutes  of  Health,  F  Building  10,  Room  10B17,  Bethesda, 

MD  20205 
POLLARD,  THOMAS  D.,  Director,  Department  of  Cell  Biology  and  Anatomy,  Johns  Hopkins 

University,  725  North  Wolfe  Street,  Baltimore,  MD  21205 

POLLOCK,  LELAND  W.,  Department  of  Zoology,  Drew  University,  Madison,  NJ  07940 
PORTER,  BEVERLY  H.,  14433  Taos  Court,  Wheaton,  MD  20906 
PORTER,  KEITH  R.,  748  Eleventh  Street,  Boulder,  CO  80302 

POTTER,  DAVID,  Department  of  Neurobiology,  Harvard  Medical  School,  Boston,  MA  021 15 
POTTER,  H.  DAVID,  Neural  Sciences  Program,  Chemistry  Building,  Indiana  University, 

Bloomington,  IN  47404 
POTTS,  WILLIAM  T.,  Department  of  Biology,  University  of  Lancaster,  Lancaster,  England, 

U.  K. 
POUSSART,  DENIS,  Department  of  Electrical  Engineering,  Universite  Laval,  Quebec,  Canada 


20  MARINE  BIOLOGICAL  LABORATORY 

PRENDERGAST,  ROBERT  A.,  Department  of  Pathology  and  Ophthalmology,  Johns  Hopkins 
University,  Baltimore,  MD  21205 

PRICE,  CARL  A.,  Waksman  Institute  of  Microbiology,  Rutgers  University,  PO  Box  759, 
Piscataway,  NJ  08854 

PRICE,  CHRISTOPHER  H.,  Biological  Science  Center,  2  Cummington  Street,  Boston,  MA 
02215 

PRIOR,  DAVID  J.,  Department  of  Biological  Sciences,  University  of  Kentucky,  Lexington, 
KY  40506 

PROSSER,  C.  LADD,  Department  of  Physiology  and  Biophysics,  Burrill  Hall  524,  University 
of  Illinois,  Urbana,  IL  61801 

PROVASOLI,  LUIGI,  Haskins  Laboratories,  165  Prospect  Street,  New  Haven,  CT  06510 

PRUSCH,  ROBERT  D.,  Department  of  Life  Sciences,  Gonzaga  University,  Spokane,  WA  99258 

PRZYBYLSKI,  RONALD  J.,  Department  of  Anatomy,  Case  Western  Reserve  University,  Cleve- 
land, OH  44104 

RABIN,  HARVEY,  PO  Box  239,  Braddock  Heights,  MD  21714 

RAMON,  FIDEL,  Department  of  Physiology,  Duke  University  Medical  Center,  Durham,  NC 
27706 

RANZI,  SILVIO,  Department  of  Zoology,  University  of  Milan,  Milan,  Italy 

RATNER,  SARAH,  Department  of  Biochemistry,  Public  Health  Research  Institute,  455  First 
Avenue,  New  York,  NY  10016 

REBHUN,  LIONEL  I.,  Department  of  Biology,  Gilmer  Hall,  University  of  Virginia,  Char- 
lottesville,  VA  22901 

REDDAN,  JOHN  R.,  Department  of  Biological  Sciences,  Oakland  University,  Rochester,  MI 
48063 

REDFIELD,  ALFRED  C.,  10  Maury  Lane,  Woods  Hole,  MA  02543 

REESE,  THOMAS  S.,  Section  on  Functional  Neuroanatomy,  National  Institutes  of  Health, 
Bethesda,  MD  20205 

REINER,  JOHN  M.,  Albany  Medical  College  of  Union  University,  Department  of  Biochem- 
istry, Albany,  NY  12208 

REINISCH,  CAROL  L.,  Tufts  University  School  of  Veterinary  Medicine,  203  Harrison  Avenue, 
Boston,  MA  02115 

REUBEN,  JOHN  P.,  Department  of  Neurology,  College  of  Physicians  and  Surgeons,  Columbia 
University,  New  York,  NY  10032 

REYNOLDS,  GEORGE  T.,  Department  of  Physics,  Jadwin  Hall,  Princeton  University, 
Princeton,  NJ  08540 

RICE,  ROBERT  V.,  Carnegie  Mellon  Institute,  4400  Fifth  Avenue,  Pittsburgh,  PA  15213 

RICKLES,  FREDERICK  R.,  University  of  Connecticut,  School  of  Medicine,  Veterans  Admin- 
istration Hospital,  Newington,  CT  061 1 1 

RIPPS,  HARRIS,  Department  of  Opthalmology,  School  of  Medicine,  New  York  University, 
550  First  Avenue,  New  York,  NY  10016 

ROBERTS,  JOHN  L.,  Department  of  Zoology,  University  of  Massachusetts,  Amherst,  MA 
01002 

ROBINSON,  DENIS  M.,  High  Voltage  Engineering  Corporation,  Burlington,  MA  01803 

ROCKSTEIN,  MORRIS,  335  Fluzia  Avenue,  Miami,  FL  33134 

RONKIN,  RAPHAEL  R.,  3212  McKinley  Street,  NW,  Washington,  DC  20015 

ROSBASH,  MICHAEL,  Rosenstiel  Basic  Medical  Research  Center,  Department  of  Biology, 
Brandeis  University,  Waltham,  MA  02154 

ROSE,  BIRGIT,  Department  of  Physiology  R-430,  School  of  Medicine,  University  of  Miami, 
PO  Box  016430,  Miami,  FL  33152 

ROSE,  S.  MERYL,  Box  309W,  Waquoit,  MA  02536 

ROSENBAUM,  JOEL  L.,  Department  of  Biology,  Kline  Biology  Tower,  Yale  University,  New 
Haven,  CT  06510 

ROSENBERG,  PHILIP,  School  of  Pharmacy,  Division  of  Pharmacology,  University  of  Con- 
necticut, Storrs,  CT  06268 

ROSENBLUTH,  JACK,  Department  of  Physiology,  School  of  Medicine,  New  York  University, 
550  First  Avenue,  New  York,  NY  10016 


MEMBERS  OF  THE  CORPORATION  21 

ROSENBLUTH,  RAJA,  3380  West  5th  Avenue,  Vancouver  8,  BC,  Canada  V6R  1R7 
ROSENKRANZ,  HERBERTS.,  Department  of  Microbiology,  New  York  Medical  College,  Val- 
halla, NY  10595 

ROSLANSKY,  JOHN,  Box  208,  Woods  Hole,  MA  02543 
ROSLANSKY,  PRISCILLA  F.,  Box  208,  Woods  Hole,  MA  02543 
Ross,  WILLIAM  N.,  Department  of  Physiology,  New  York  Medical  College,  Valhalla,  NY 

10595 
ROTH,  JAY  S.,  Division  of  Biological  Sciences,  Section  of  Biochemistry  and  Biophysics, 

University  of  Connecticut,  Storrs,  CT  06268 
ROWE,  DOROTHY,  88  Chestnut  Hill,  Boston,  MA  02165 

ROWLAND,  LEWIS  P.,  Neurological  Institute,  710  West  168th  Street,  New  York,  NY  10032 
RUDERMAN,  JOAN  V.,  Department  of  Anatomy,  Harvard  Medical  School,  Boston,  MA  02 1 1 5 
RUSHFORTH,  NORMAN  B.,  Department  of  Biology,  Case  Western  Reserve  University,  Cleve- 
land, OH  44106 
RUSSELL-HUNTER,  W.  D.,  Department  of  Biology,  110  Lyman  Hall,  Syracuse  University, 

Syracuse,  NY  13210 
RUSTAD,  RONALD  C.,  Radiology  Department,  Case  Western  Reserve  University,  Cleveland, 

OH  44106 

SAGER,  RUTH,  Sidney  Farber  Cancer  Institute,  44  Binney  Street,  Boston,  MA  02115 
SALAMA,  GUY,  Department  of  Physiology,  University  of  Pittsburgh,  Pittsburgh,  PA  15261 
SALMON,  EDWARD  D.,  Department  of  Zoology,  University  of  North  Carolina,  Chapel  Hill, 

NC  27514 
SALZBERG,  BRIAN  M.,  Department  of  Physiology,  University  of  Pennsylvania,  4010  Locust 

Street,  Philadelphia,  PA  19174 

SANDERS,  HOWARD,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  MA  02543 
SANGER,  JEAN  M.,  Department  of  Anatomy,  School  of  Medicine,  University  of  Pennsylvania, 

36th  and  Hamilton  Walk,  Philadelphia,  PA  19174 
SANGER,  JOSEPH,  Department  of  Anatomy,  School  of  Medicine,  University  of  Pennsylvania, 

36th  and  Hamilton  Walk,  Philadelphia,  PA  19174 
SATO,  HIDEMI,  Sugashima  Marine  Biological  Laboratory,  Nagoya  University,  Sugashima- 

cho,  Toba-shi,  Mie-Ken  517,  Japan 

SAUNDERS,  JOHN,  JR.,  Department  of  Biological  Sciences,  SUNY,  Albany,  NY  12222 
SAZ,  ARTHUR  K.,  Medical  and  Dental  Schools,  Georgetown  University,  3900  Reservoir 

Road,  NW,  Washington,  DC  20051 
SCHACHMAN,  HOWARD  K.,  Department  of  Molecular  Biology,  University  of  California, 

Berkeley,  CA  94720 
SCHIFF,  JEROME  A.,  Institute  for  Photobiology  of  Cells  and  Organelles,  Brandeis  University, 

Waltham,  MA  02154 
SCHLESINGER,  R.  WALTER,  Department  of  Microbiology,  College  of  Medicine  and  Dentistry, 

Rutgers  University,  PO  Box  101,  Piscataway,  NJ  08854 
SCHMEER,  SISTER  ARLINEC.,  American  Cancer  Research  Center  and  Hospital,  6401  West 

Colfax  Avenue,  Denver,  CO  80214 
SCHNEIDERMAN,  HOWARD  K.,  Monsanto  Company,  800  North  Lindberg  Blvd.  (D1W),  St. 

Louis,  MO  63166 
SCHOPF,  THOMAS  J.  M.,  Department  of  Geophysical  Sciences,  University  of  Chicago,  5734 

South  Ellis  Avenue,  Chicago,  IL  60637 

SCHOTTE,  OSCAR  E.,  Department  of  Biology,  Amherst  College,  Amherst,  MA  01002 
SCHUEL,  HERBERT,  Department  of  Anatomical  Sciences,  SUNY,  Buffalo,  NY  14214 
SCHUETZ,  ALLEN  W.,  School  of  Hygiene  and  Public  Health,  Johns  Hopkins  University, 

Baltimore,  MD  21205 
SCHWAB,  WALTER  E.,  Department  of  Biology,  Virginia  Polytechnical  Institute  and  State 

University,  Blacksburg,  VA  24601 
SCHWARTZ,  TOBIAS  L.,  Biological  Sciences  Group,  University  of  Connecticut,  Storrs,  CT 

06268 

SCOTT,  ALLAN  C.,  1  Nudd  Street,  Waterville,  ME  04901 
SCOTT,  GEORGE  T.,  10  Orchard  Street,  Woods  Hole,  MA  02543 


22  MARINE  BIOLOGICAL  LABORATORY 

SEARS,  MARY,  PO  Box  152,  Woods  Hole,  MA  02543 

SEGAL,  SHELDON  J.,  Director,  Population  Division,  The  Rockefeller  Foundation,  1133  Av- 
enue of  the  Americas,  New  York,  NY  10036 

SELIGER,  HOWARD  H.,  Johns  Hopkins  University,  McCollum-Pratt  Institute,  Baltimore,  MD 
21218 

SELMAN,  KELLY,  Department  of  Anatomy,  College  of  Medicine,  University  of  Florida, 
Gainesville,  FL  32601 

SENFT,  JOSEPH,  Rodale  Research  Center,  Box  323,  RD1,  Kutztown,  PA  19530 

SHANKLIN,  DOUGLAS  R.,  PO  Box  1267,  Gainesville,  FL  32602 

SHAPIRO,  HERBERT,  6025  North  13th  Street,  Philadelphia,  PA  19141 

SHAVER,  GAIUS  R.,  Ecosystems  Center,  Marine  Biological  Laboratory,  Woods  Hole,  MA 
02543 

SHAVER,  JOHN  R.,  Department  of  Zoology,  Michigan  State  University,  E.  Lansing,  MI 
48823 

SHEPARD,  DAVID  C,  PO  Box  44,  Woods  Hole,  MA  02543 

SHEPRO,  DAVID,  Department  of  Biology,  Boston  University,  2  Cummington  Street,  Boston, 
MA  02215 

SHERMAN,  I.  W.,  Division  of  Life  Sciences,  University  of  California,  Riverside,  CA  92502 

SHILO,  MOSHE,  Head,  Department  of  Microbiological  Chemistry,  Hebrew  University,  Je- 
rusalem, Israel 

SHOUKIMAS,  JONATHAN  J.,  Laboratory  of  Biophysics,  NINCDS,  Marine  Biological  Labo- 
ratory, Woods  Hole,  MA  02543 

SHRIVASTAV,  BRIJ  S.,  Department  of  Pharmacology,  Duke  University  Medical  Center,  Dur- 
ham, NC  27710 

SIEGEL,  IRWIN  M.,  Department  of  Ophthalmology,  Medical  Center,  New  York  University, 
550  First  Avenue,  New  York,  NY  10016 

SIEGELMAN,  HAROLD  W.,  Department  of  Biology,  Brookhaven  National  Laboratory,  Upton, 
NY  11973 

SIMON,  ERIC  J.,  New  York  University,  Medical  School,  550  First  Avenue,  New  York,  NY 
10016 

SJODIN,  RAYMOND  A.,  Department  of  Biophysics,  University  of  Maryland,  Baltimore,  MD 
21201 

SKINNER,  DOROTHY  M.,  Biology  Division,  Oak  Ridge  National  Laboratory,  Oak  Ridge,  TN 
37830 

SLOBODKIN,  LAWRENCE  B.,  Department  of  Biology,  SUNY,  Stony  Brook,  NY  1 1790 

SMITH,  HOMER  P.,  General  Manager,  Marine  Biological  Laboratory,  Woods  Hole,  MA 
02543 

SMITH,  MICHAEL  A.,  Foreign  and  Commonwealth  Office,  King  Charles  Street,  London 
SW1A  2AH,  England,  U.  K. 

SMITH,  PAUL  F.,  PO  Box  264,  Woods  Hole,  MA  02543 

SMITH,  RALPH  I.,  Department  of  Zoology,  University  of  California,  Berkeley,  CA  94720 

SORENSON,  ALBERT  L.,  Department  of  Physiology,  Albert  Einstein  College  of  Medicine, 
1300  Morris  Park  Avenue,  Bronx,  NY  10461 

SORENSON,  MARTHA  M.,  Department  of  Neurology,  College  of  Physicians  and  Surgeons, 
Columbia  University,  New  York,  NY  10032 

SPECK,  WILLIAM  T.,  Department  of  Pediatrics,  Case  Western  Reserve  University,  Cleveland, 
OH  44106 

SPECTOR,  A.,  College  of  Physicians  and  Surgeons,  Black  Bldg.  Room  1516,  Columbia  Uni- 
versity, New  York,  NY  10032 

SPIEGEL,  EVELYN,  Department  of  Biological  Sciences,  Dartmouth  College,  Hanover,  NH 
02755 

SPIEGEL,  MELVIN,  Department  of  Biological  Sciences,  Dartmouth  College,  Hanover,  NH 
02755 

SPRAY,  DAVID  C.,  Department  of  Neurosciences,  Albert  Einstein  College  of  Medicine,  1300 
Morris  Park  Avenue,  Bronx,  NY  10461 

STARZAK,  MICHAEL  E.,  Department  of  Chemistry,  SUNY,  Binghamton,  NY  13901 


MEMBERS  OF  THE  CORPORATION  23 

STEELE,  JOHN  Hyslop,  Director,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  MA 
02543 

STEINACHER,  ANTOINETTE,  Department  of  Biophysics,  The  Rockefeller  University,  New 
York,  NY  10021 

STEINBERG,  MALCOLM,  Department  of  Biology,  Princeton  University,  Princeton,  NJ  08540 

STEPHENS,  GROVER  C.,  Department  of  Developmental  and  Cell  Biology,  University  of  Cal- 
ifornia, Irvine,  CA  92717 

STEPHENS,  RAYMOND  E.,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

STETTEN,  DEWITT,  JR.,  Senior  Scientific  Advisor,  National  Institutes  of  Health,  Building 
16,  Room  118,  Bethesda,  MD  20205 

STETTEN,  MARJORIE  R.,  National  Institutes  of  Health,  Bldg.  10,  9B-02,  Bethesda,  MD 
20205 

STOKES,  DARRELL  R.,  Department  of  Biology,  Emory  University,  Atlanta,  GA  30322 

STRACHER,  ALFRED,  Downstate  Medical  Center,  SUNY,  450  Clarkson  Avenue,  Brooklyn, 
NY  11203 

STREHLER,  BERNARD  L.,  2235  25th  Street,  #217,  San  Pedro,  CA  90732 

STUART,  ANN  E.,  Medical  Sciences  Research  Wing  206H,  Department  of  Physiology,  Uni- 
versity of  North  Carolina,  Chapel  Hill,  NC  27514 

SUMMERS,  WILLIAM  C.,  Huxley  College,  Western  Washington  State  College,  Bellingham, 
WA  98225 

SUSSMAN,  MAURICE,  Department  of  Life  Sciences,  University  of  Pittsburgh,  Pittsburgh,  PA 
15260 

SWENSON,  RANDOLPHEP.,  JR.,  Department  of  Physiology  G-4,  University  of  Pennsylvania, 
Philadelphia,  PA  19174 

SZABO,  GEORGE,  Harvard  School  of  Dental  Medicine,  188  Longwood  Avenue,  Boston,  MA 
02115 

SZAMIER,  R.  BRUCE,  Harvard  Medical  School,  Berman-Gund  Laboratory,  Massachusetts 
Eye  and  Ear  Infirmary,  243  Charles  Street,  Boston,  MA  021 14 

SZENT-GYORGYI,  ALBERT,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

SZENT-GYORGYI,  ANDREW,  Department  of  Biology,  Brandeis  University,  Waltham,  MA 
02154 

TAKASHIMA,  SHIRO,  Department  of  Bioengineering,  University  of  Pennsylvania,  Philadel- 
phia, PA  19174 

TAMM,  SIDNEY  L.,  Boston  University  Marine  Program,  Marine  Biological  Laboratory, 
Woods  Hole,  MA  02543 

TANZER,  MARVIN  L.,  Department  of  Biochemistry,  Box  G,  Medical  School,  University  of 
Connecticut,  Farmington,  CT  06032 

TASAK.I,  ICHIJI,  Laboratory  of  Neurobiology,  NIMH,  National  Institutes  of  Health,  Be- 
thesda, MD  20205 

TAYLOR,  DOUGLASS  L.,  The  Biological  Laboratories,  Harvard  University,  Cambridge,  MA 
02138 

TAYLOR,  ROBERT  E.,  Laboratory  of  Biophysics,  NINCDS,  National  Institutes  of  Health, 
Bethesda,  MD  20205 

TAYLOR,  W.  ROWLAND,  1540  Northbourne  Road,  Baltimore,  MD  21239 

TELFER,  WILLIAM  H.,  Department  of  Biology,  University  of  Pennsylvania,  Philadelphia,  PA 
19174 

THORNDIKE,  W.  NICHOLAS,  Wellington  Management  Company,  28  State  Street,  Boston, 
MA  02109 

TIFFNEY,  WESLEY  N.,  226  Edge  Hill  Road,  Sharon,  MA  02067 

TRACER,  WILLIAM,  The  Rockefeller  University,  66th  Street  and  York  Avenue,  New  York, 
NY  10021 

TRAVIS,  D.  M.,  Veterans  Administration  Medical  Center,  Fargo,  ND  58102 

TREISTMAN,  STEVEN  N.,  Worcester  Foundation  for  Experimental  Biology,  Shrewsbury,  MA 
01545 

TRINKAUS,  J.  PHILIP,  Osborn  Zoological  Labs,  Department  of  Zoology,  Yale  University, 
New  Haven,  CT  06510 


24  MARINE  BIOLOGICAL  LABORATORY 

TROLL,  WALTER,  Department  of  Environmental  Medicine,  College  of  Medicine,  New  York 
University,  550  First  Avenue,  New  York,  NY  10016 

TROXLER,  ROBERT  F.,  Department  of  Biochemistry,  School  of  Medicine,  Boston  University, 
80  East  Concord  Street,  Boston,  MA  02118 

TURNER,  RUTH  D.,  Mollusk  Department,  Museum  of  Comparative  Zoology,  Harvard  Uni- 
versity, Cambridge,  MA  02138 

TWEEDELL,  KENYON  S.,  Department  of  Biology,  University  of  Notre  Dame,  Notre  Dame, 
IN  46656 

URETZ,  ROBERT  B.,  Division  of  Biological  Sciences,  University  of  Chicago,  950  East  59th 
Street,  Chicago,  IL  60637 

VALIELA,  IVAN,  Boston  University  Marine  Program,  Marine  Biological  Laboratory,  Woods 
Hole,  MA  02543 

VALOIS,  JOHN,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

VAN  HOLDE,  KENSAL,  Department  of  Biochemistry  and  Biophysics,  Oregon  State  University, 
Corvallis,  OR  97331 

VILLEE,  CLAUDE  A.,  Department  of  Biological  Chemistry,  Harvard  Medical  School,  Boston, 
MA  02115 

VINCENT,  WALTER  S.,  School  of  Life  and  Health  Sciences,  University  of  Delaware,  Newark, 
DE  19711 

WAINIO,  WALTER,  W.,  Box  1059,  Nelson  Labs,  Rutgers  Biochemistry,  Piscataway,  NJ  08854 

WAKSMAN,  BYRON,  National  Multiple  Sclerosis  Society,  205  East  42nd  Street,  New  York, 
NY  10017 

WALKER,  CHARLES  A.,  3113  Shamrock  South,  Tallahassee,  FL  32303 

WALL,  BETTY,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

WALLACE,  ROBIN  A.,  Department  of  Anatomy,  College  of  Medicine,  University  of  Florida, 
Gainesville,  FL  32610 

WANG,  AN,  Bedford  Road,  Lincoln,  MA  01773 

WARNER,  ROBERT  C.,  Department  of  Molecular  Biology  and  Biochemistry,  University  of 
California,  Irvine,  CA  92717 

WARREN,  KENNETH  S.,  The  Rockefeller  Foundation,  1133  Avenue  of  the  Americas,  New 
York,  NY  10036 

WARREN,  LEONARD,  Department  of  Therapeutic  Research,  School  of  Medicine,  Anatomy- 
Chemistry  Building  Room  337,  University  of  Pennsylvania,  Philadelphia,  PA  19174 

WATERMAN,  T.  H.,  Yale  University,  610  Kline  Biology  Tower,  New  Haven,  CT  06510 

WATSON,  STANLEY,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  MA  02543 

WEBB,  H.  MARGUERITE,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

WEBER,  ANNEMARIE,  Department  of  Biochemistry,  School  of  Medicine,  University  of  Penn- 
sylvania, Philadelphia,  PA  19174 

WEBSTER,  FERRIS,  800  25th  Street,  NW,  Washington,  DC  20037 

WEIDNER,  EARL,  Department  of  Zoology  and  Physiology,  Louisiana  State  University,  Baton 
Rouge,  LA  70803 

WEISS,  LEON,  P.,  Department  of  Animal  Biology,  School  of  Veterinary  Medicine,  University 
of  Pennsylvania,  Philadelphia,  PA  19174 

WEISSMAN,  GERALD,  New  York  University,  550  First  Avenue,  New  York,  NY  10016 

WERMAN,  ROBERT,  Neurobiology  Unit,  The  Hebrew  University,  Jerusalem,  Israel 

WESTERFIELD,  R.  MONTE,  The  Institute  of  Neuroscience,  University  of  Oregon,  Eugene, 
OR  97403 

WHITTAKER,  J.  RICHARD,  Director,  Boston  University  Marine  Program,  Marine  Biological 
Laboratory,  Woods  Hole,  MA  02543 

WIERCINSK.I,  FLOYD  J.,  Department  of  Biology,  Northeastern  Illinois  University,  5500  North 
St.  Louis  Avenue,  Chicago,  IL  60625 

WIGLEY,  ROLAND  L.,  35  Wilson  Road,  Woods  Hole,  MA  02543 

WILBER,  CHARLES  G.,  Department  of  Zoology,  Colorado  State  University,  Fort  Collins,  CO 
80523 

WILSON,  DARCY  B.,  Department  of  Pathology,  School  of  Medicine,  University  of  Pennsyl- 
vania, Philadelphia,  PA  19174 

WILSON,  EDWARD  O.,  Department  of  Zoology,  Harvard  University,  Cambridge,  MA  02138 


MEMBERS  OF  THE  CORPORATION 


25 


WILSON,  T.  HASTINGS,  Department  of  Physiology,  Harvard  Medical  School,  Boston    MA 

02115 

WILSON,  WALTER  L.,  Department  of  Biology,  Oakland  University,  Rochester,  MI  48063 
WITKOVSKY,  PAUL,  Department  of  Ophthalmology,  New  York  University  Medical  Center, 

550  First  Avenue,  New  York,  NY  10016 
WITTENBERG,  JONATHAN  B.,  Department  of  Physiology  and  Biochemistry,  Albert  Einstein 

College  of  Medicine,  1300  Morris  Park  Avenue,  Bronx,  NY  10461 
WOELKERLING,  WILLIAM  J.,  Department  of  Botany,  Latrobe  University,  Bundoora,  Victoria, 

Australia  3083 
WOLF,  DON  P.,  Department  of  OB-GYN,  University  of  Texas  Health  Sciences  Center,  6431 

Fannin,  Houston,  TX  77030 
WOODWELL,  GEORGE  M.,  Director,  Ecosystems  Center,  Marine  Biological  Laboratory, 

Woods  Hole,  MA  02543 
WORGUL,  BASIL  V.,  Department  of  Ophthalmology,  Columbia  University,  630  W.  168th  St., 

New  York,  NY  10032 
Wu,   CHAU   HSIUNG,   Department  of  Pharmacology,   Northwestern   University   Medical 

School,  Chicago,  I L  6061 1 
WYTTENBACH,  CHARLES  R.,  Department  of  Physiology  and  Cell  Biology,  University  of 

Kansas,  Lawrence,  KS  06045 

YAMIN,  MICHAEL  A.,  The  Rockefeller  University,  1230  York  Avenue,  New  York,  NY  10021 
YEH,  JAY  Z.,  Department  of  Pharmacology,  Northwestern  University  Medical  School,  303 

E.  Chicago  Avenue,  Chicago,  IL  6061 1 

YOUNG,  RICHARD,  100  Royalston  Road,  Wellesley  Hills,  MA  02181 

YPHANTIS,  DAVID  A.,  Department  of  Biochemistry  and  Biophysics,  University  of  Connec- 
ticut, Storrs,  CT  06268 
ZIGMAN,  SEYMOUR,  School  of  Medicine  and  Dentistry,  University  of  Rochester,  260  Crit- 

tenden  Boulevard,  Rochester,  NY  14620 
ZIMMERMAN,  A.  M.,  Department  of  Zoology,  University  of  Toronto,  Toronto  5,  Ontario, 

Canada 
ZUCKER,  ROBERT  S.,  Department  of  Physiology,  University  of  California,  Berkeley,  CA 

94720 

ASSOCIATE  MEMBERS 


ACKROYD,  DR.  AND  MRS.  FREDERICK  W. 

ADELBERG,  DR.  AND  MRS.  EDWARD  A. 
ADELMAN,  DR.  AND  MRS.  WILLIAM  J. 
AHEARN,  MR.  AND  MRS.  DAVID  C. 
ALLEN,  Miss  CAMILLA  K. 
ALLEN,  DRS.  ROBERT  D.  AND  NINA  S. 
AMBERSON,  MRS.  WILLIAM  R. 
ANDERSON,  DRS.  JAMES  L.  AND  HELENE  M. 

ARMSTRONG,  DR.  AND  MRS.  SAMUEL  C. 

ARNOLD,  DR.  AND  MRS.  JOHN 
ATWOOD,  DR.  AND  MRS.  KIMBALL  C. 
BALL,  MRS.  ERIC  G. 
BALLANTINE,  DR.  AND  MRS.  H.  T.,  JR. 
BANG,  MRS.  FREDERIK  B. 
BANKS,  MR.  AND  MRS.  W.  L. 
BARROWS,  MRS.  ALBERT  W. 
BENNETT,  DR.  AND  MRS.  MICHAEL  V.  L. 
BERNHEIMER,  DR.  ALAN  W. 
BERNSTEIN,  MR.  AND  MRS.  NORMAN 
BIGELOW,  MRS.  ROBERT  O. 
BLACKBURN,  DR.  AND  MRS.  GEORGE  L. 

BODEEN,  MR.  AND  MRS.  GEORGE  H. 
BOETTIGER,  DR.  AND  MRS.  EDWARD  G. 
BOLTON,  MR.  AND  MRS.  THOMAS  C. 


BORGESE,  DR.  AND  MRS.  THOMAS  A. 

BOTKIN,  DR.  DANIEL  B. 
BOWLES,  DR.  AND  MRS.  FRANCIS  P. 
BRADLEY,  DR.  AND  MRS.  CHARLES  C. 
BRONSON,  MRS.  SAMUEL  C. 
BROWN,  MRS.  DUGALD  E.  S. 
BROWN,  DR.  AND  MRS.  F.  A.,  JR. 
BROWN,  DR.  AND  MRS.  THORNTON 
BUCK,  MRS.  JOHN  B. 

BUFFINGTON,  MRS.  ALICE  H. 
BUFFINGTON,  MRS.  GEORGE 

BURGER,  DR.  AND  MRS.  MAX  M. 
BURROUGH,  MRS.  ARNOLD  H. 
BURT,  MR.  AND  MRS.  CHARLES  E. 
BUTLER,  MRS.  E.  G. 
BUTLER,  MR.  AND  MRS.  RHETT  W. 
CALKINS,  MR.  AND  MRS.  G.  N.,  JR. 
CAMPBELL,  DR.  AND  MRS.  DAVID  G. 
CAMPBELL,  MR.  AND  MRS.  WORTHINGTON, 

JR. 

CAPOBIANCO,  MR.  AND  MRS.  PAT  J. 
CARLSON,  DR.  AND  MRS.  FRANCIS 
CARLTON,  MR.  AND  MRS.  WINSLOW  G. 
CASHMAN,  MR.  AND  MRS.  EUGENE  R. 


26 


MARINE  BIOLOGICAL  LABORATORY 


CHAMBERS,  DR.  AND  MRS.  EDWARD  L. 
CHENEY,  DR.  AND  MRS.  RALPH  H. 
CLAFF,  MR.  AND  MRS.  MARK 
CLARK,  MR.  AND  MRS.  HAYS 
CLARK,  MRS.  JAMES  McC. 
CLARK,  DR.  AND  MRS.  LEONARD  B. 
CLARK,  MR.  AND  MRS.  LEROY,  JR. 
CLARK,  MRS.  W.  VAN  ALAN 
CLEMENT,  DR.  AND  MRS.  A.  C. 
CLOWES  FUND,  INC. 

CLOWES,  DR.  AND  MRS.  ALEXANDER  W. 
CLOWES,  MR.  ALLEN  W. 
CLOWES,  DR.  AND  MRS.  G.  H.  A.,  JR. 
COHEN,  DR.  AND  MRS.  SEYMOUR 
COLEMAN,  DR.  AND  MRS.  JOHN 
CONNELL,  MR.  AND  MRS.  W.  J. 
COOPER,  MR.  AND  MRS.  JOHN  H.,  JR. 
COPELAND,  MRS.  D.  EUGENE 

COPELAND,  MR.  AND  MRS.  PRESTON  S. 

COSTELLO,  MRS.  DONALD  P. 
GRAIN,  MR.  AND  MRS.  MELVIN  C. 
CRAMER,  MR.  AND  MRS.  IAN  D.  W. 
CRANE,  MR.  JOHN 
CRANE,  JOSEPHINE,  FOUNDATION 
CRANE,  MRS.  W.  CAREY 
CROSS,  MR.  AND  MRS.  NORMAN  C. 
CROSSLEY,  MR.  AND  MRS.  ARCHIBALD  M. 
CROWELL,  DR.  AND  MRS.  SEARS 
DAIGNAULT,  MR.  AND  MRS.  A.  T. 
DANIELS,  MR.  AND  MRS.  BRUCE  G. 
DAY,  MR.  AND  MRS.  POMEROY 
DICKSON,  DR.  WILLIAM  A. 
DRUMMOND,  MR.  AND  MRS.  A.  H.,  JR. 
DuBois,  DR.  AND  MRS.  A.  B. 

DUNKERLEY,  MR.  AND  MRS.   H.  GORDON 

DUPONT,  MR.  A.  FELIX,  JR. 
DYER,  MR.  AND  MRS.  ARNOLD  W. 
EBERT,  DR.  AND  MRS.  JAMES  D. 
EDWARDS,  DR.  AND  MRS.  ROBERT  L. 
EGLOFF,  DR.  AND  MRS.  F.  R.  L. 
ELLIOTT,  MRS.  ALFRED  M. 
ELSMITH,  MRS.  DOROTHY  O. 
EPPEL,  MR.  AND  MRS.  DUDLEY 
EVANS,  MR.  AND  MRS.  DUDLEY 
EWING,  DR.  AND  MRS.  GIFFORD  C. 
FENNO,  MRS.  EDWARD  N. 
FERGUSON,  DR.  AND  MRS.  J.  J.,  JR. 
FINE,  DR.  AND  MRS.  JACOB 
FISHER,  MRS.  B.  C. 
FISHER,  MR.  FREDERICK  S.,  Ill 
FISHER,  DR.  AND  MRS.  SAUL  H. 
FRANCIS,  MR.  AND  MRS.  LEWIS  W.,  JR. 
FRIES,  DR.  AND  MRS.  E.  F.  B. 
FYE,  DR.  AND  MRS.  PAUL  M. 
GABRIEL,  DR.  AND  MRS.  MORDECAI  L. 
GAISER,  DR.  AND  MRS.  DAVID  W. 
GARFIELD,  Miss  ELEANOR 


GARREY,  DR.  AND  MRS.  WALTER 
GELLIS,  DR.  AND  MRS.  SYDNEY 
GERMAN,  DR.  AND  MRS.  JAMES  L.,  Ill 
GIFFORD,  MR.  AND  MRS.  JOHN  A. 
GIFFORD,  DR.  AND  MRS.  PROSSER 
GILBERT,  DR.  AND  MRS.  DANIEL  L. 
GILBERT,  MRS.  HELEN  H. 
GILDEA,  DR.  MARGARET  C.  L. 
GILLETTE,  MR.  AND  MRS.  ROBERT  S. 
GLASS,  DR.  AND  MRS.  H.  BENTLEY 
GLAZEBROOK,  MRS.  JAMES  R. 
GLUSMAN,  DR.  AND  MRS.  MURRAY 
GOLDMAN,  DR.  AND  MRS.  ALLEN  S. 
GOLDSTEIN,  MRS.  MOISE  H.,  JR. 
GRANT,  DR.  AND  MRS.  PHILIP 
GRASSLE,  MR.  AND  MRS.  J.  K. 
GREEN,  Miss  GLADYS  M. 
GREENE,  MR.  AND  MRS.  WILLIAM  C. 
GREER,  MR.  AND  MRS.  W.  H.,  JR. 
GROSCH,  DR.  AND  MRS.  DANIEL  S. 
GROSS,  MRS.  PAUL  R. 

GRUSON,  MRS.  MARTHA  C. 

GUNNING,  MR.  AND  MRS.  ROBERT 

HAIGH,  MR.  AND  MRS.  RICHARD  H. 

HALVORSON,  DR.  AND  MRS.  HARLYN  O. 

HANDLER,  MRS.  PHILIP 

HARVEY,  DR.  AND  MRS.  RICHARD  B. 

HASSETT,  DR.  AND  MRS.  CHARLES 

HASTINGS,  MRS.  J.  WOODLAND 

HEFFRON,  DR.  AND  MRS.  RODERICK 

HENLEY,  DR.  CATHERINE 

HIAM,  MR.  AND  MRS.  E.  W. 

HIATT,  DR.  AND  MRS.  HOWARD 

HILL,  MRS.  SAMUEL  E. 

HlLSINGER,  MR.  AND  MRS.  ARTHUR 

HlRSCHFELD,  MRS.  NATHAN  B. 

HOBBIE,  DR.  AND  MRS.  JOHN 

HOCKER,  MR.  AND  MRS.  LON 

HOFFMAN,  REV.  AND  MRS.  CHARLES 

HORWITZ,  DR.  AND  MRS.  NORMAN  H. 

HOUSTON,  MR.  AND  MRS.  HOWARD  E. 

HUETTNER,  DR.  AND  MRS.  ROBERT 
HUNZIKER,  MR.  AND  MRS.   HERBERT  E. 

HYNES,  MR.  AND  MRS.  THOMAS  J.,  JR. 

INOUE,  MRS.  SHINYA 

IRELAND,  MRS.  HERBERT  A. 

ISSOKSON,  MR.  AND  MRS.  ISRAEL 

IVENS,  DR.  SUE 

JACKSON,  Miss  ELIZABETH  B. 

JANNEY,  MRS.  WISTAR 

JEWETT,  G.  F.,  FOUNDATION 

JEWETT,  MR.  AND  MRS.  G.  F.,  JR. 

JONES,  MR.  AND  MRS.  DEWITT,  III 

JONES,  MR.  AND  MRS.  FREDERICK,  III 

JORDAN,  DR.  AND  MRS.  EDWIN  P. 

KAAN,  DR.  HELEN  W. 

KAHLER,  MR.  AND  MRS.  GEORGE  A. 


MEMBERS  OF  THE  CORPORATION 


27 


KAHLER,  MR.  AND  MRS.  ROBERT  W. 
KAMINER,  DR.  AND  MRS.  BENJAMIN 
KARUSH,  DR.  AND  MRS.  FRED 
KEITH,  MRS.  JEAN  R. 
KELLEHER,  MR.  AND  MRS.  PAUL  R. 
KEOSIAN,  MRS.  JESSIE 

KlEN,  MR.  AND  MRS.  PlETER 

KINNARD,  MRS.  L.  RICHARD 
KIVY,  DR.  AND  MRS.  PETER 
KOHN,  DR.  AND  MRS.  HENRY  I. 

KOLLER,  DR.  AND  MRS.  LEWIS  R. 

KUFFLER,  MRS.  STEPHEN  W. 
LADERMAN,  MR.  AND  MRS.  EZRA 
LASH,  DR.  AND  MRS.  JAMES 
LASTER,  DR.  AND  MRS.  LEONARD 
LAUFER,  DR.  AND  MRS.  HANS 
LAVIGNE,  MARGARET  M. 
LAWRENCE,  MR.  FREDERICK  V. 
LAWRENCE,  MRS.  WILLIAM 
LAZAROW,  MRS.  ARNOLD 
LEATHERBEE,  MR.  JOHN  A. 
LEMANN,  MRS.  LUCY  B. 
LENHER,  MR.  AND  MRS.  SAMUEL 
LEVINE,  DR.  AND  MRS.  RACHMIEL 
LEWIS,  MR.  JOHN  T. 
LITTLE,  MRS.  ELBERT 
LOEB,  MRS.  ROBERT  F. 

LOVELL,  MR.  AND  MRS.   HOLLIS  R. 

LOWE,  DR.  AND  MRS.  CHARLES  W. 

LOWENGARD,  MRS.  JOSEPH 

MACKEY,  MR.  AND  MRS.  WILLIAM  K. 
MACLEISH,  MRS.  MARGARET 
MACNARY,  MR.  B.  GLENN 
MAcNicHOL,  DR.  AND  MRS.  EDWARD  F., 

JR. 

MAHER,  Miss  ANNE  CAMILLE 
MARKS,  DR.  AND  MRS.  PAUL  A. 
MARSLAND,  DR.  AND  MRS.  DOUGLAS 
MARTYNA,  MR.  AND  MRS.  JOSEPH 
MARVIN,  DR.  DOROTHY  H. 
MASER,  DR.  AND  MRS.  MORTON 
MASTROIANNI,  DR.  AND  MRS.  L.,  JR. 
MATHER,  MR.  AND  MRS.  FRANK  J.,  Ill 
MATTHIESSEN,  MR.  AND  MRS.  G.  C. 

MCCUSKER,  MR.  AND  MRS.  PAUL  T. 

MCELROY,  MRS.  NELLA  W. 
MCLANE,  MRS.  T.  THORNE 
MEIGS,  MR.  AND  MRS.  ARTHUR 
MEIGS,  DR.  AND  MRS.  J.  WISTER 
MELILLO,  DR.  AND  MRS.  JERRY 
MELLON,  RICHARD  KING,  FOUNDATION 
MELLON,  MR.  AND  MRS.  RICHARD  P. 
MENKE,  DR.  W.  J. 
METZ,  MRS.  CHARLES  B. 
MEYERS,  MR.  AND  MRS.  RICHARD 
MILLER,  DR.  DANIEL  A. 
MIXTER,  MR.  AND  MRS.  W.  J.,  JR. 


MONTGOMERY,  DR.  AND  MRS.  CHARLES  H. 
MONTGOMERY,  MR.  AND  MRS.  RAYMOND 

P. 

MORSE,  MR.  AND  MRS.  CHARLES  L.,  JR. 
MORSE,  MR.  AND  MRS.  RICHARD  S. 
MOUL,  MRS.  EDWIN  T. 
NEWTON,  C.  H.,  BUILDERS,  INC. 
NICHOLS,  MRS.  GEORGE 
NlCKERSON,  MR.  AND  MRS.  FRANK  L. 
NORMAN,  MR.  AND  MRS.  ANDREW  E. 
NORMAN  FOUNDATION 
O'HERRON,  MR.  AND  MRS.  JONATHAN 
O'SULLIVAN,  DR.  RENEE  BENNETT 
ORTINS,  MR.  ARMAND 
PALMER,  MRS.  DOUGLAS  W. 
PAPPAS,  DR.  AND  MRS.  GEORGE  D. 
PARK,  MRS.  FRANKLIN  A. 
PARK,  MR.  AND  MRS.  MALCOLM  S. 
PARMENTER,  Miss  CAROLYN  L. 
PARMENTIER,  MR.  GEORGE  L. 
PENDELTON,  DR.  AND  MRS.  MURRAY  E. 
PENDERGAST,  MRS.  CLAUDIA 
PENNINGTON,  Miss  ANNE  H. 
PERKINS,  MR.  AND  MRS.  COURTLAND  D. 
PERSON,  DR.  AND  MRS.  PHILIP 
PETERSON,  MR.  AND  MRS.  E.  GUNNAR 
PETERSON,  MR.  AND  MRS.  E.  JOEL 
PETERSON,  MR.  RAYMOND  W. 
PHILIPPE,  MR.  AND  MRS.  PIERRE 
PORTER,  DR.  AND  MRS.  KEITH  R. 
PROSSER,  MRS.  C.  LADD 
PUTNAM,  MR.  ALLAN  RAY 
PUTNAM,  MR.  AND  MRS.  W.  A.,  Ill 
PYNE,  Miss  RUTH 
RAYMOND,  DR.  AND  MRS.  SAMUEL 
READ,  Ms.  LEE 

REDFIELD,  DR.  AND  MRS.  ALFRED  C. 
RENEK,  MR.  AND  MRS.  MORRIS 
REYNOLDS,  DR.  AND  MRS.  GEORGE 
REYNOLDS,  MRS.  BARTOW 
REZNIKOFF,  DR.  AND  MRS.  PAUL 
RICCA,  DR.  AND  MRS.  RENATO  A. 
RIGGS,  MR.  AND  MRS.  LAWRASSON,  III 
RIINA,  MR.  AND  MRS.  JOHN  R. 
ROBB,  Ms.  ALISON  A. 
ROBERTSON,  MRS.  C.  STUART 
ROBERTSON,  DR.  AND  MRS.  C.  W. 
ROBINSON,  DR.  AND  MRS.  DENIS  M. 
ROGERS,  MRS.  JULIAN 
ROOT,  MRS.  WALTER  S. 
Ross,  DR.  VIRGINIA 
ROWE,  MRS.  WILLIAM  S. 
RUBIN,  DR.  JOSEPH 
RUGH,  MRS.  ROBERTS 
RUSSELL,  MR.  AND  MRS.  HENRY  D. 
RYDER,  MR.  AND  MRS.  FRANCIS  C. 
SAUNDERS,  DR.  AND  MRS.  JOHN  W. 


28 


MARINE  BIOLOGICAL  LABORATORY 


SAUNDERS,  MRS.  LAWRENCE 
SAWYER,  MR.  AND  MRS.  JOHN  E. 

SCHLESINGER,  MRS.   R.  WALTER 

SCOTT,  MRS.  GEORGE  T. 

SCOTT,  MRS.  NORMAN  E. 

SEARS,  MR.  AND  MRS.  HAROLD  B. 

SEGAL,  DR.  AND  MRS.  SHELDON  J. 

SHAPIRO,  MRS.  HARRIET  S. 

SHEMIN,  DR.  AND  MRS.  DAVID 

SHEPRO,  DR.  AND  MRS.  DAVID 

SMITH,  MRS.  HOMER  P. 

SMITH,  MR.  VANDORN  C. 

SNIDER,  MR.  ELIOT 

SPECHT,  MRS.  HEINZ 

SPIEGEL,  DR.  AND  MRS.  MELVIN 

STEELE,  MRS.  M.  EVELYN 

STEINBACH,  MRS.  H.  B. 

STETTEN,  DR.  AND  MRS.  DE\VITT,  JR. 

STRACHER,  DR.  AND  MRS.  ALFRED 

STUNKARD,  DR.  HORACE 

STURTEVANT,  MRS.  A.  H. 

SWANSON,  DR.  AND  MRS.  CARL  P. 

SWOPE,  MR.  AND  MRS.  GERARD  L. 
SWOPE,  MRS.  GERARD,  JR. 
TAYLOR,  MARJORIE  G. 
TIETJE,  MR.  AND  MRS.  EMIL  D.,  JR. 
TODD,  MR.  AND  MRS.  GORDON  F. 

TOLKAN,  MR.  AND  MRS.  NORMAN  N. 


TOMPKINS,  MRS.  B.  A. 
TRACER,  MRS.  WILLIAM 
TROLL,  DR.  AND  MRS.  WALTER 
TULLY,  MR.  AND  MRS.  GORDON  F. 
VALOIS,  MR.  AND  MRS.  JOHN 
VAN  BRUNT,  MR.  AND  MRS.  A.  H.,  JR. 
VEEDER,  MRS.  RONALD  A. 
WAITE,  MR.  AND  MRS.  CHARLES  E. 
WAKSMAN,  DR.  AND  MRS.  BYRON  H. 
WARE,  MR.  AND  MRS.  J.  LINDSAY 
WARREN,  MRS.  SHIELDS 
WATT,  MR.  AND  MRS.  JOHN  B. 
WEISBERG,  MR.  AND  MRS.  ALFRED  M. 
WHEATLEY,  DR.  MARJORIE  A. 
WHEELER,  DR.  AND  MRS.  PAULS. 

AND  MRS.  RALPH  E. 

AND  MRS.  GEOFFREY  G.,  JR. 

WlCHTERMAN,  DR.  AND  MRS.  RALPH 
WlCKERSHAM,  MR.  AND  MRS.  A.  A.  TlLNEY 
WlCKERSHAM,  MRS.  JAMES  H.,  JR. 

WILHELM,  DR.  HAZEL  S. 
WlTMER,  DR.  AND  MRS.  ENOS  E. 
WOLFINSOHN,  MR.  AND  MRS.  WOLFE 
WOODWELL,  MRS.  GEORGE 
YNTEMA,  MRS.  CHESTER  L. 
ZINN,  DR.  AND  MRS.  DONALD  J. 
ZIPF,  DR.  ELIZABETH 
ZWILLING,  MRS.  EDGAR 


WHEELER,  DR. 
WHITNEY,  MR. 


III.  CERTIFICATE  OF  ORGANIZATION 

(On  File  in  the  Office  of  the  Secretary  of  the  Commonwealth) 


No.  3170 


We,  Alpheus  Hyatt,  President,  William  Stanford  Stevens,  Treasurer,  and  William  T.  Sedg- 
wick,  Edward  G.  Gardiner,  Susan  Mims  and  Charles  Sedgwick  Minot  being  a  majority  of 
the  Trustees  of  the  Marine  Biological  Laboratory  in  compliance  with  the  requirements  of 
the  fourth  section  of  chapter  one  hundred  and  fifteen  of  the  Public  Statutes  do  hereby  certify 
that  the  following  is  a  true  copy  of  the  agreement  of  association  to  constitute  said  Corporation, 
with  the  names  of  the  subscribers  thereto:- 

We,  whose  names  are  hereto  subscribed,  do,  by  this  agreement,  associate  ourselves  with  the 
intention  to  constitute  a  Corporation  according  to  the  provisions  of  the  one  hundred  and 
fifteenth  chapter  of  the  Public  Statutes  of  the  Commonwealth  of  Massachusetts,  and  the 
Acts  in  amendment  thereof  and  in  addition  thereto. 

The  name  by  which  the  Corporation  shall  be  known  is  THE  MARINE  BIOLOGICAL 
LABORATORY. 


The  purpose  for  which  the  Corporation  is  constituted  is  to  establish  and  maintain  a  laboratory 
or  station  for  scientific  study  and  investigations,  and  a  school  for  instruction  in  biology  and 
natural  history. 


CERTIFICATE  OF  ORGANIZATION  29 

The  place  within  which  the  Corporation  is  established  or  located  is  the  city  of  Boston  within 
said  Commonwealth. 

The  amount  of  its  capital  stock  is  none. 

In  Witness  Whereof,  we  have  hereunto  set  our  hands,  this  twenty  seventh  day  of  February 
in  the  year  eighteen  hundred  and  eighty-eight,  Alpheus  Hyatt,  Samuel  Mills,  William  T. 
Sedgwick,  Edward  G.  Gardiner,  Charles  Sedgwick  Minot,  William  G.  Farlow,  William  Stan- 
ford Stevens,  Anna  D.  Phillips,  Susan  Mims,  B.  H.  Van  Vleck. 

That  the  first  meeting  of  the  subscribers  to  said  agreement  was  held  on  the  thirteenth  day 
of  March  in  the  year  eighteen  hundred  and  eighty-eight. 

In  Witness  Whereof,  we  have  hereunto  signed  our  names,  this  thirteenth  day  of  March  in 
the  year  eighteen  hundred  and  eighty-eight,  Alpheus  Hyatt,  President,  William  Stanford 
Stevens,  Treasurer,  Edward  G.  Gardiner,  William  T.  Sedgwick,  Susan  Mims,  Charles  Sedg- 
wick Minot. 

(Approved  on  March  20,  1888  as  follows: 

/  hereby  certify  that  it  appears  upon  an  examination  of  the  within  written  certificate  and  the 
records  of  the  corporation  duly  submitted  to  my  inspection,  that  the  requirements  of  sections 
one,  two  and  three  of  chapter  one  hundred  and  fifteen,  and  sections  eighteen,  twenty  and 
twenty-one  of  chapter  one  hundred  and  six,  of  the  Public  Statutes,  have  been  complied  with 
and  I  hereby  approve  said  certificate  this  twentieth  day  of  March  A.D.  eighteen  hundred 
and  eighty-eight. 

CHARLES  ENDICOTT 
Commissioner  of  Corporations) 


IV.  ARTICLES  OF  AMENDMENT 

(On  File  in  the  Office  of  the  Secretary  of  the  Commonwealth) 

We,  James  D.  Ebert,  President,  and  David  Shepro,  Clerk  of  the  Marine  Biological  Laboratory, 
located  at  Woods  Hole,  Massachusetts  02543,  do  hereby  certify  that  the  following  amendment 
to  the  Articles  of  Organization  of  the  Corporation  was  duly  adopted  at  a  meeting  held  on 
August  15,  1975,  as  adjourned  to  August  29,  1975,  by  vote  of  444  members,  being  at  least 
two-thirds  of  its  members  legally  qualified  to  vote  in  the  meetings  of  the  corporation: 

VOTED:  That  the  Certificate  of  Organization  of  this  corporation  be  and  it  hereby  is 
amended  by  the  addition  of  the  following  provisions: 

"No  Officer,  Trustee  or  Corporate  Member  of  the  corporation  shall  be  personally 
liable  for  the  payment  or  satisfaction  of  any  obligation  or  liabilities  incurred  as 
a  result  of,  or  otherwise  in  connection  with,  any  commitments,  agreements, 
activities  or  affairs  of  the  corporation. 

"Except  as  otherwise  specifically  provided  by  the  Bylaws  of  the  corporation, 
meetings  of  the  Corporate  Members  of  the  corporation  may  be  held  anywhere 
in  the  United  States. 


30  MARINE  BIOLOGICAL  LABORATORY 

"The  Trustees  of  the  corporation  may  make,  amend  or  repeal  the  Bylaws  of  the 
corporation  in  whole  or  in  part,  except  with  respect  to  any  provisions  thereof 
which  shall  by  law,  this  Certificate  or  the  Bylaws  of  the  corporation,  require 
action  by  the  Corporate  Members." 

The  foregoing  amendment  will  become  effective  when  these  articles  of  amendment  are  filed 
in  accordance  with  Chapter  180,  Section  7  of  the  General  Laws  unless  these  articles  specify, 
in  accordance  with  the  vote  adopting  the  amendment,  a  later  effective  date  not  more  than 
thirty  days  after  such  filing,  in  which  event  the  amendment  will  become  effective  on  such 
later  date. 

In  Witness  whereof  and  Under  the  Penalties  of  Perjury,  we  have  hereto  signed  our  names 
this  2nd  day  of  September,  in  the  year  1975,  James  D.  Ebert,  President;  David  Shepro,  Clerk. 

(Approved  on  October  24,  1975,  as  follows: 

I  hereby  approve  the  within  articles  of  amendment  and,  the  filing  fee  in  the  amount  of  $10 

having  been  paid,  said  articles  are  deemed  to  have  been  filed  with  me  this  24th  day  of  October, 

1975. 

PAUL  GUZZI 

Secretary  of  the  Commonwealth) 

V.  BYLAWS  OF  THE  CORPORATION  OF  THE  MARINE 
BIOLOGICAL  LABORATORY 

(Revised  August  11,  1978) 

I.  (A)  The  name  of  the  Corporation  shall  be  The  Marine  Biological  Laboratory.  The 
Corporation's  purpose  shall  be  to  establish  and  maintain  a  laboratory  or  station  for  scientific 
study  and  investigation,  and  a  school  for  instruction  in  biology  and  natural  history. 

(B)  Marine  Biological  Laboratory  admits  students  without  regard  to  race,  color,  sex, 
national  and  ethnic  origin  to  all  the  rights,  privileges,  programs  and  activities  generally 
accorded  or  made  available  to  students  in  its  courses.  It  does  not  discriminate  on  the  basis 
of  race,  color,  sex,  national  and  ethnic  origin  in  employment,  administration  of  its  educational 
policies,  admissions  policies,  scholarship  and  other  programs. 

II.  (A)  The  members  of  the  Corporation  ("Members")  shall  consist  of  persons  elected 
by  the  Board  of  Trustees,  upon  such  terms  and  conditions  and  in  accordance  with  such 
procedures,  not  inconsistent  with  law  or  these  Bylaws,  as  may  be  determined  by  said  Board 
of  Trustees.  Except  as  provided  below,  any  Member  may  vote  at  any  meeting,  either  in  person 
or  by  proxy  executed  no  more  than  six  months  prior  to  the  date  of  such  meeting.  Members 
shall  serve  until  their  death  or  resignation  unless  earlier  removed,  with  or  without  cause,  by 
the  affirmative  vote  of  two-thirds  of  the  Trustees  then  in  office.  Any  member  who  has  attained 
the  age  of  seventy  years  or  has  retired  from  his  home  institution  shall  automatically  be 
designated  a  Life  Member  provided  he  signifies  his  wish  to  retain  his  membership.  Life 
Members  shall  not  have  the  right  to  vote  and  shall  not  be  assessed  for  dues. 

(B)  The  Associates  of  the  Marine  Biological  Laboratory  shall  be  an  unincorporated  group 
of  persons  (including  associations  and  corporations)  interested  in  the  Laboratory  and  shall 
be  organized  and  operated  under  the  general  supervision  and  authority  of  the  Trustees. 

III.  The  officers  of  the  Corporation  shall  consist  of  a  Chairman  of  the  Board  of  Trustees, 
President,  Director,  Treasurer  and  Clerk,  elected  or  appointed  by  the  Trustees  as  set  forth 
in  Article  IX. 


BYLAWS  31 

IV.  The  Annual  Meeting  of  the  Members  shall  be  held  on  the  Friday  following  the 
Second  Tuesday  in  August  in  each  year  at  the  Laboratory  in  Woods  Hole,  Massachusetts, 
at  9:30  a.m.  Subject  to  the  provisions  of  Article  VIII(2),  at  such  meeting  the  Members  shall 
choose  by  ballot  six  Trustees  to  serve  four  years,  and  shall  transact  such  other  business  as 
may  properly  come  before  the  meeting.  Special  meetings  of  the  Members  may  be  called  by 
the  Chairman  or  Trustees  to  be  held  at  such  time  and  place  as  may  be  designated. 

V.  Twenty  five  Members  shall  constitute  a  quorum  at  any  meeting.  Except  as  otherwise 
required  by  law  or  these  Bylaws,  the  affirmative  vote  of  a  majority  of  the  Members  voting 
in  person  or  by  proxy  at  a  meeting  attended  by  a  quorum  (present  in  person  or  by  proxy) 
shall  constitute  action  on  behalf  of  the  Members. 

VI.  (A)  Inasmuch  as  the  time  and  place  of  the  Annual  Meeting  of  Members  are  fixed 
by  these  Bylaws,  no  notice  of  the  Annual  Meeting  need  be  given.  Notice  of  any  special 
meeting  of  Members,  however,  shall  be  given  by  the  Clerk  by  mailing  notice  of  the  time  and 
place  and  purpose  of  such  meeting,  at  least  15  days  before  such  meeting,  to  each  Member 
at  his  or  her  address  as  shown  on  the  records  of  the  Corporation. 

(B)  Any  meeting  of  the  Members  may  be  adjourned  to  any  other  time  and  place  by  the 
vote  of  a  majority  of  those  Members  present  or  represented  at  the  meeting,  whether  or  not 
such  Members  constitute  a  quorum.  It  shall  not  be  necessary  to  notify  any  Member  of  any 
adjournment. 

VII.  The  Annual  Meeting  of  the  Trustees  shall  be  held  promptly  after  the  Annual  Meet- 
ing of  the  Corporation  at  the  Laboratory  in  Woods  Hole,  Massachusetts.  Special  meetings 
of  the  Trustees  shall  be  called  by  the  Chairman,  the  President,  or  by  any  seven  Trustees,  to 
be  held  at  such  time  and  place  as  may  be  designated.  Notice  of  Trustees'  meetings  may  be 
given  orally,  by  telephone,  telegraph  or  in  writing;  and  notice  given  in  time  to  enable  the 
Trustees  to  attend,  or  in  any  case  notice  sent  by  mail  or  telegraph  to  a  Trustee's  usual  or 
last  known  place  or  residence,  at  least  one  week  before  the  meeting  shall  be  sufficient.  Notice 
of  a  meeting  need  not  be  given  to  any  Trustee  if  a  written  waiver  of  notice,  executed  by  him 
before  or  after  the  meeting  is  filed  with  the  records  of  the  meeting,  or  if  he  shall  attend  the 
meeting  without  protesting  prior  thereto  or  at  its  commencement  the  lack  of  notice  to  him. 

VIII.  (A)  There  shall  be  four  groups  of  Trustees: 

(1)  Trustees  (the  "Corporate  Trustees")  elected  by  the  Members  according  to  such  pro- 
cedures, not  inconsistent  with  these  Bylaws,  as  the  Trustees  shall  have  determined.  Except 
as  provided  below,  such  Trustees  shall  be  divided  into  four  classes  of  six,  one  class  to  be 
elected  each  year  to  serve  for  a  term  of  four  years.  Such  classes  shall  be  designated  by  the 
year  of  expiration  of  their  respective  terms. 

(2)  Trustees  ("Board  Trustees")  elected  by  the  Trustees  then  in  office  according  to  such 
procedures,  not  inconsistent  with  these  Bylaws,  as  the  Trustees  shall  have  determined.  Except 
as  provided  below,  such  Board  Trustees  shall  be  divided  into  four  classes  of  three,  one  class 
to  be  elected  each  year  to  serve  for  a  term  of  four  years.  Such  classes  shall  be  designated 
by  the  year  of  expiration  of  their  respective  terms.  It  is  contemplated  that,  unless  otherwise 
determined  by  the  Trustees  for  good  reason,  Board  Trustees  shall  be  individuals  who  have 
not  been  considered  for  election  as  Corporate  Trustees. 

(3)  Trustees  ex  officio,  who  shall  be  the  Chairman,  the  President,  the  Director,  the 
Treasurer,  and  the  Clerk. 

(4)  Trustees  emeriti  who  shall  include  any  Member  who  has  attained  the  age  of  seventy 
years  (or  the  age  of  sixty  five  and  has  retired  from  his  home  institution)  and  who  has  served 
a  full  elected  term  as  a  regular  Trustee,  provided  he  signifies  his  wish  to  serve  the  Laboratory 
in  that  capacity.  Any  Trustee  who  qualifies  for  emeritus  status  shall  continue  to  serve  as  a 


32  MARINE  BIOLOGICAL  LABORATORY 

regular  Trustee  until  the  next  Annual  Meeting  whereupon  his  office  as  regular  Trustee  shall 
become  vacant  and  be  filled  by  election  by  the  Members  or  by  the  Board,  as  the  case  may 
be.  The  Trustees  ex  officio  and  emeriti  shall  have  all  the  rights  of  the  Trustees,  except  that 
Trustees  emeriti  shall  not  have  the  right  to  vote. 

(B)  The  aggregate  number  of  Corporate  Trustees  and  Board  Trustees  elected  in  any  year 
(excluding  Trustees  elected  to  fill  vacancies  which  do  not  result  from  expiration  of  a  term) 
shall  not  exceed  nine.  The  number  of  Board  Trustees  so  elected  shall  not  exceed  three  and 
unless  otherwise  determined  by  vote  of  the  Trustees,  the  number  of  Corporate  Trustees  so 
elected  shall  not  exceed  ;  ix. 

(C)  The  Trustees  and  Officers  shall  hold  their  respective  offices  until  their  successors  are 
chosen  in  their  stead. 

(D)  Any  Trustee  may  be  removed  from  office  at  any  time  with  or  without  cause,  by  vote 
of  a  majority  of  the  Members  entitled  to  vote  in  the  election  of  Trustees;  or  for  cause,  by 
vote  of  two-thirds  of  the  Trustees  then  in  office.  A  Trustee  may  be  removed  for  cause  only 
if  notice  of  such  action  shall  have  been  given  to  all  of  the  Trustees  or  Members  entitled  to 
vote,  as  the  case  may  be,  prior  to  the  meeting  at  which  such  action  is  to  be  taken  and  if  the 
Trustee  so  to  be  removed  shall  have  been  given  reasonable  notice  and  opportunity  to  be  heard 
before  the  body  proposing  to  remove  him. 

(E)  Any  vacancy  in  the  number  of  Corporate  Trustees,  however  arising,  may  be  filled 
by  the  Trustees  then  in  office  unless  and  until  filled  by  the  Members  at  the  next  Annual 
Meeting.  Any  vacancy  in  the  number  of  Board  Trustees  may  be  filled  by  the  Trustees. 

(F)  A  Corporate  Trustee  or  a  Board  Trustee  who  has  served  an  initial  term  of  at  least 
2  years  duration  shall  be  eligible  for  re-election  to  a  second  term,  but  shall  be  ineligible  for 
re-election  to  any  subsequent  term  until  two  years  have  elapsed  after  he  last  served  as  Trustee. 

IX.  (A)  The  Trustees  shall  have  the  control  and  management  of  the  affairs  of  the  Cor- 
poration. They  shall  elect  a  Chairman  of  the  Board  of  Trustees  who  shall  be  elected  annually 
and  shall  serve  until  his  successor  is  selected  and  qualified  and  who  shall  also  preside  at 
meetings  of  the  Corporation.  They  shall  elect  a  President  of  the  Corporation  who  shall  also 
be  the  Vice  Chairman  of  the  Board  of  Trustees  and  Vice  Chairman  of  meetings  of  the 
Corporation,  and  who  shall  be  elected  annually  and  shall  serve  until  his  successor  is  selected 
and  qualified.  They  shall  annually  elect  a  Treasurer  who  shall  serve  until  his  successor  is 
selected  and  qualified.  They  shall  elect  a  Clerk  (a  resident  of  Massachusetts)  who  shall  serve 
for  a  term  of  4  years.  Eligibility  for  re-election  shall  be  in  accordance  with  the  content  of 
Article  VIII  (F)  as  applied  to  Corporate  or  Board  Trustees.  They  shall  elect  Board  Trustees 
as  described  in  Article  VIII  (B).  They  shall  appoint  a  Director  of  the  Laboratory  for  a  term 
not  to  exceed  five  years,  provided  the  term  shall  not  exceed  one  year  if  the  candidate  has 
attained  the  age  of  65  years  prior  to  the  date  of  the  appointment.  They  may  choose  such 
other  officers  and  agents  as  they  may  think  best.  They  may  fix  the  compensation  and  define 
the  duties  of  all  the  officers  and  agents  of  the  Corporation  and  may  remove  them  at  any 
time.  They  may  fill  vacancies  occurring  in  any  of  the  offices.  The  Board  of  Trustees  shall 
have  the  power  to  choose  an  Executive  Committee  from  their  own  number  as  provided  in 
Article  X,  and  to  delegate  to  such  Committee  such  of  their  own  powers  as  they  may  deem 
expedient  in  addition  to  those  powers  conferred  by  Article  X.  They  shall  from  time  to  time 
elect  Members  to  the  Corporation  upon  such  terms  and  conditions  as  they  shall  have  deter- 
mined, not  inconsistent  with  law  or  these  Bylaws. 

( B)  The  Board  of  Trustees  shall  also  have  the  power,  by  vote  of  a  majority  of  the  Trustees 
then  in  Office,  to  elect  an  Investment  Committee  and  any  other  committee  and,  by  like  vote, 
to  delegate  thereto  some  or  all  of  their  powers  except  those  which  by  law,  the  Articles  of 
Organization  or  these  Bylaws  they  are  prohibited  from  delegating.  The  members  of  any  such 
committee  shall  have  such  tenure  and  duties  as  the  Trustees  shall  determine;  provided  that 
the  Investment  Committee,  which  shall  oversee  the  management  of  the  Corporation's  en- 
dowment funds  and  marketable  securities,  shall  include  the  Chairman  of  the  Board  of 
Trustees,  the  Treasurer  of  the  Corporation,  and  the  Chairman  of  the  Corporation's  Budget 
Committee,  as  ex  officio  members,  together  with  such  Trustees  as  may  be  required  for  not 


BYLAWS  33 

less  than  two-thirds  of  the  Investment  Committee  to  consist  of  Trustees.  Except  as  otherwise 
provided  by  these  Bylaws  or  determined  by  the  Trustees,  any  such  committee  may  make 
rules  for  the  conduct  of  its  business;  but,  unless  otherwise  provided  by  the  Trustees  or  in 
such  rules,  its  business  shall  be  conducted  as  nearly  as  possible  in  the  same  manner  as  is 
provided  by  these  Bylaws  for  the  Trustees. 

X.  (A)  The  Executive  Committee  is  hereby  designated  to  consist  of  not  more  than  ten 
members,  including  the  ex  officio  Members  (Chairman  of  the  Board  of  Trustees,  President, 
Director  and  Treasurer);  and  six  additional  Trustees,  two  of  whom  shall  be  elected  by  the 
Board  of  Trustees  each  year,  to  serve  for  a  three-year  term. 

(B)  The  Chairman  of  the  Board  of  Trustees  shall  act  as  Chairman  of  the  Executive 
Committee,  and  the  President  as  Vice  Chairman.  A  majority  of  the  members  of  the  Executive 
Committee  shall  constitute  a  quorum  and  the  affirmative  vote  of  a  majority  of  those  voting 
at  any  meeting  at  which  a  quorum  is  present  shall  constitute  action  on  behalf  of  the  Executive 
Committee.  The  Executive  Committee  shall  meet  at  such  times  and  places  and  upon  such 
notice  and  appoint  such  sub-committees  as  the  Committee  shall  determine. 

(C)  The  Executive  Committee  shall  have  and  may  exercise  all  the  powers  of  the  Board 
during  the  intervals  between  meetings  of  the  Board  of  Trustees  except  those  powers  specif- 
ically withheld  from  time  to  time  by  vote  of  the  Board  or  by  law.  The  Executive  Committee 
may  also  appoint  such  committees,  including  persons  who  are  not  Trustees,  as  it  may  from 
time  to  time  approve  to  make  recommendations  with  respect  to  matters  to  be  acted  upon  by 
the  Executive  Committee  or  the  Board  of  Trustees. 

(D)  The  Executive  Committee  shall  keep  appropriate  minutes  of  its  meetings  and  its 
action  shall  be  reported  to  the  Board  of  Trustees. 

(E)  The  elected  Members  of  the  Executive  Committee  shall  constitute  as  a  standing 
"Committee  for  the  Nomination  of  Officers,"  responsible  for  making  nominations,  at  each 
Annual  Meeting  of  the  Corporation,  and  of  the  Board  of  Trustees,  for  candidates  to  fill  each 
office  as  the  respective  terms  of  office  expire  (Chairman  of  the  Board,  President,  Director, 
Treasurer,  and  Clerk). 

XI.  A  majority  of  the  Trustees,  the  Executive  Committee,  or  any  other  committee  elected 
by  the  Trustees  shall  constitute  a  quorum;  and  a  lesser  number  than  a  quorum  may  adjourn 
any  meeting  from  time  to  time  without  further  notice.  At  any  meeting  of  the  Trustees,  the 
Executive  Committee,  or  any  other  committee  elected  by  the  Trustees,  the  vote  of  a  majority 
of  those  present,  or  such  different  vote  as  may  be  specified  by  law,  the  Articles  of  Organization 
or  these  Bylaws,  shall  be  sufficient  to  take  any  action. 

XII.  Any  action  required  or  permitted  to  be  taken  at  any  meeting  of  the  Trustees,  the 
Executive  Committee  or  any  other  committee  elected  by  the  Trustees  as  referred  to  under 
Article  IX  may  be  taken  without  a  meeting  if  all  of  the  Trustees  or  members  of  such 
committee,  as  the  case  may  be,  consent  to  the  action  in  writing  and  such  written  consents 
are  filed  with  the  records  of  meetings.  The  Trustees  or  members  of  the  Executive  Committee 
or  any  other  committee  appointed  by  the  Trustees  may  also  participate  in  meeting  by  means 
of  conference  telephone,  or  otherwise  take  action  in  such  a  manner  as  may  from  time  to  time 
be  permitted  by  law. 

XIII.  The  consent  of  every  Trustee  shall  be  necessary  to  dissolution  of  the  Marine  Bi- 
ological Laboratory.  In  case  of  dissolution,  the  property  shall  be  disposed  of  in  such  manner 
and  upon  such  terms  as  shall  be  determined  by  the  affirmative  vote  of  two-thirds  of  the  Board 
of  Trustees  then  in  office. 

XIV.  These  Bylaws  may  be  amended  by  the  affirmative  vote  of  the  Members  at  any 
meeting,  provided  that  notice  of  the  substance  of  the  proposed  amendment  is  stated  in  the 


34  MARINE  BIOLOGICAL  LABORATORY 

notice  of  such  meeting.  As  authorized  by  the  Articles  of  Organization,  the  Trustees,  by  a 
majority  of  their  number  then  in  office,  may  also  make,  amend,  or  repeal  these  Bylaws,  in 
whole  or  in  part,  except  with  respect  to  (a)  the  provisions  of  these  Bylaws  governing  (i)  the 
removal  of  Trustees  and  (ii)  the  amendment  of  these  Bylaws  and  (b)  any  provisions  of  these 
Bylaws  which  by  law,  the  Articles  of  Organization  or  these  Bylaws,  requires  action  by  the 
Members. 

No  later  than  the  time  of  giving  notice  of  the  meeting  of  Members  next  following  the 
making,  amending  or  repealing  by  the  Trustees  of  any  Bylaw,  notice  thereof  stating  the 
substance  of  such  change  shall  be  given  to  all  Corporation  Members  entitled  to  vote  on 
amending  the  Bylaws. 

Any  Bylaw  adopted  by  the  Trustees  may  be  amended  or  repealed  by  the  Members  entitled 
to  vote  on  amending  the  Bylaws. 

XV.  The  account  of  the  Treasurer  shall  be  audited  annually  by  a  certified  public  ac- 
countant. 

XVI.  The  Corporation  will  indemnify  every  person  who  is  or  was  a  trustee,  officer  or 
employee  of  the  Corporation  or  a  person  who  provides  services  without  compensation  to  an 
Employee  Benefit  Plan  maintained  by  the  Corporation,  for  any  liability  (including  reasonable 
costs  of  defense  and  settlement )  arising  by  reason  of  any  act  or  omission  affecting  an  Employee 
Benefit  Plan  maintained  by  the  Corporation  or  affecting  the  participants  or  beneficiaries  of 
such  Plan,  including  without  limitation  any  damages,  civil  penalty  or  excise  tax  imposed 
pursuant  to  the  Employee  Retirement  Income  Security  Act  of  1974;  provided,  (1)  that  the 
Act  or  omission  shall  have  occurred  in  the  course  of  the  person's  service  as  trustee  or  officer 
of  the  Corporation  or  within  the  scope  of  the  employment  of  an  employee  of  the  Corporation 
or  in  connection  with  a  service  provided  without  compensation  to  an  Employee  Benefit  Plan 
maintained  by  the  Corporation,  (2)  that  the  Act  or  omission  be  in  good  faith  as  determined 
by  the  Corporation  (whose  determination  made  in  good  faith  and  not  arbitrarily  or  capri- 
ciously shall  be  conclusive),  and  (3)  that  the  Corporation's  obligation  hereunder  shall  be 
offset  to  the  extent  of  any  otherwise  applicable  insurance  coverage,  under  a  policy  maintained 
by  the  Corporation  or  any  other  person,  or  other  source  of  indemnification. 

VI.  REPORT  OF  THE  DIRECTOR 

Introduction 

The  fashion  of  substituting  for  the  traditional  Christmas  card  a  lengthy  circular 
letter,  often  ill-duplicated  and  partially  illegible,  continues  to  gain  devotees.  These 
Director's  Reports,  like  circular  letters,  are  supposed  to  highlight  events  of  the  year 
past.  I  wish  that  they,  too,  had  a  deadline  for  composition  close  to  the  New  Year. 
Then  I  should  have  before  me  several  fresh  examples,  just  arrived,  of  the  circular 
Christmas  letter.  Some  would  be  hortatory,  but  some  would  show  me  how  not  to 
proceed. 

"Do  not,"  they  would  warn,  "try  to  cover  everything  that  happened  in  the  course 
of  the  year.  Do  not,  as  a  slovenly  alternative,  deal  only  with  what  interests  you, 
or  solely  with  what  has  happened  during  the  past  two  weeks.  Do  not  assume  that 
the  recipients  share  your  self-pity  or  self-aggrandizement.  Do  not  select,  from 
among  all  the  subject  possibilities,  an  undisciplined  few." 

What  not  to  do  is,  unfortunately,  easier  here  as  elsewhere  to  exemplify  than 
is  the  positive.  If  the  life  of  a  small  family  over  the  course  of  a  year  is  unyielding 
to  epistolary  rules,  then  how  intractable  is  a  year's  life  of  a  great  institution,  if  it 
must  be  encompassed  within  a  report  of  a  few  pages  that  are  less  than  instantly 
soporific!  How  much  more  intractable  still  is  such  a  year's  life  when  it  has  been 


REPORT  OF  THE  DIRECTOR  35 

marked  by  problems  and  progress,  defeats  and  achievements,  hopes  proven  vain 
and  hopes  fulfilled! 

Still,  like  the  correspondent  who  needed,  last  Christmas,  to  tell  us  the  year's 
truth  of  her  family  in  four  pages  (single-spaced)  devoted  to  her  husband's  lower- 
back  pain,  I  feel  duty-bound  to  try.  I,  too  am  a  neighbor  to  psychogenic  discomforts. 
Having  just  now  discovered  her  manuscript,  mimeographed  on  green-tinted  bond 
paper,  amidst  the  schematics  for  our  TV  (which  faltered  at  Christmas-time),  I  am 
newly  mindful  of  the  pitfalls  of  the  genre,  and  of  the  laughter  implicit  in  it.  I  have 
approached  my  task  seriously  enough  this  year,  knowing  that  it  will  be  judged  dull 
or  sharp  according  to  the  reader's  preconceptions.  But  I  have  done  the  thing  with 
a  light  heart,  keeping  before  me  a  specific  application  of  the  great  truth  discovered 
by  Will  Rogers:  "It's  no  trick  being  a  humorist  when  you  have  the  whole  government 
working  for  you." 

Think,  then,  of  these  few  pages  as  a  kind  of  MBL-family  Christmas  letter; 
arriving,  not  with  the  ice-crust  that  rims  the  Eel  Pond  in  December,  but  with  the 
mid-August  rafts  of  cruising  boats  there.  Think  of  it  as  an  attempt  to  provide  the 
absent  member  with  some  sense  of  what  has  happened  since  the  last  issue;  but  not 
with  a  comprehensive  and  representative  summary  of  all  the  toothaches,  the  raffle- 
prizes,  the  IRS  refund,  the  reports  from  teacher  about  our  youngest's  being  cheeky 
again.  There  is  a  real  comfort  if  the  attempt  fails:  all  the  facts  are  summarized 
comprehensively  in  the  remaining,  and  far  more  important,  pages  of  this  Annual 
Report.  The  financial  ones  carry  the  imprimatur,  not  only  of  our  indispensable 
Treasurer,  but  also  of  our  admirably  scrupulous  auditors. 

Construction  and  Rehabilitation:  Capital  Campaign  Phase  I 

By  the  time  this  report  is  printed  and  distributed,  all  but  a  few  minor  components 
of  the  Phase  I  projects,  planned  and  announced  as  the  first  steps  of  our  Second 
Century  Fund  campaign,  will  have  been  funded  and  completed.  The  Environmental 
Sciences  Center  and  the  Candle  House  restoration  were  completed  during  1981: 
Rehabilitation  of  Lillie  began  very  early  in  1982,  that  project  having  been  fully 
subscribed  during  the  prior  year.  Most  of  it  will  be  done  by  June.  The  final  jobs 
await  a  quieter  time  after  Labor  Day,  when  certain  specialized  equipment — such 
as  the  replacement  for  Lillie's  wonderful  elevator — will  be  delivered,  and  when 
there  is  once  again  the  possibility  that  our  contractor  may  park  his  large  vehicles, 
trailer-trucks,  and  cranes  behind  Lillie  without  causing  an  outbreak  of  violence. 

The  Environmental  Sciences  Center  has  turned  out  far  better  than  we  dared 
to  hope.  The  architects  (Peirce,  Pierce  &  Kramer)  have  done  an  imaginative  job 
in  an  exceedingly  difficult  assignment:  to  convert  an  old,  frame-and-shingle  dor- 
mitory building  to  attractive  office,  conference,  and  teaching  space,  and  then  to 
attach  the  result,  in  an  aesthetically  acceptable  way,  to  a  modern,  high-technology 
laboratory  annex.  All  that  has  been  done  without  compromising  harmony  of  style 
and  scale  on  the  Quadrangle,  and  without  ruining  the  view  toward  Great  Harbor. 

As  good-looking  as  the  facility  is,  it  is  also  practical.  All  staff  of  the  Ecosystems 
Center  now  occupy  it,  and  it  serves  their  purposes  efficiently.  There  remains  a 
significant  amount  of  unfinished  laboratory  space  that  will  provide  for  rational 
expansion  and  probably  for  transient  use.  The  Environmental  Sciences  Center  is 
a  great  step  forward  for  MBL  ecology.  As  is  always  the  case  in  new  laboratory 
buildings,  minor  problems  have  surfaced  with  heavy  utilization  of  the  facilities,  but 
those  are  reparable  and  will  soon  be  corrected. 


36  MARINE  BIOLOGICAL  LABORATORY 

The  Candle  House  will  surely  be  a  model  for  similar  restorations  elsewhere. 
No  important  feature  of  its  external  appearance  has  been  altered,  but  the  interior 
is  entirely  new,  and  entirely  satisfactory.  Here,  too  the  architects  (Earl  R.  Flans- 
burgh  and  Associates)  combined  sensitivity  and  good  taste  with  technical  skill,  to 
bring  a  splendid  old  building,  long  dead  so  far  as  habitability  was  concerned, 
completely  back  to  life.  The  administration  are  now  housed  there  and  have  been 
since  January,  1982.  Unless  my  eyes  and  ears  deceive  me,  they  are  housed  in 
comfort  and  with  decent  furnishings  consistent  with  the  importance  of  their  jobs. 
All  of  the  space  cleared  in  Lillie  has  become  new  laboratories  or  the  expanded 
domain  of  the  MBL  Library. 

Details  of  the  Lillie  rehabilitation,  which  is  a  project  more  costly  and  far  more 
complex  than  the  others  of  Phase  I,  are  properly  left  to  the  Annual  Report  for 
1982,  since  the  bulk  of  the  work  will  have  been  done  in  that  year.  1981  was, 
nevertheless,  the  year  that  saw  this  undertaking,  keystone  of  the  entire  plan  for 
campus  rehabilitation,  funded.  The  Kresge  Foundation's  challenge  grant  ($0.5 
million)  was  paid  in  recognition  of  our  having  raised  another  million  (and  somewhat 
more)  in  direct  grants  and  pledges,  including  one  of  $100,000  from  the  MBL 
Associates  in  aid  of  the  Auditorium  renovations. 

The  donors  to  all  these  projects  are  too  numerous  to  mention  here.  The  gratitude 
owed  them  is  too  great  to  be  expressed  properly  in  a  circular  letter,  but  it  is  proper 
to  note  that  all  parts  of  the  private  sector  were  represented:  Corporations,  large 
and  modest-sized;  charitable  Foundations;  individuals,  within  and  without  the  im- 
mediate MBL  family,  the  gifts  from  those  good  people  covering  the  entire  range 
of  possibilities,  from  bequests  of  real  property  to  large,  outright  gifts  of  cash. 

The  work  of  Phase  I  will  have  cost,  in  toto,  more  than  was  planned  in  1979, 
but  by  the  standard  of  similar  undertakings  in  other  institutions,  we  have  done 
remarkably  well  in  these  three  inflation-plagued  years.  More  importantly,  the  fund 
raising  effort  has  accomplished  more  than  simply  to  stay  on  schedule:  it  has  in  fact 
kept  pace,  overall,  with  the  actual  costs  of  the  work.  As  will  be  evident  in  a  later 
section  of  this  Report,  the  MBL  financial  staff  deserve  special  thanks  for  managing 
their  part  of  this  intricate  undertaking. 

As  to  the  fund-raising  itself,  there  is  not  much  more  to  be  said:  it  has  kept  pace. 
We  could,  and  should,  however,  have  done  even  better.  The  MBL's  message  is,  as 
I  know  from  experience,  quite  unique.  That  makes  our  case,  once  the  chance  to 
present  it  comes,  differentiable  from  that  of  the  host  of  schools,  colleges,  and  social 
agencies  now  clamoring  for  the  attention  of  private  philanthropy.  The  clamor  is, 
moreover,  increasing  steadily  as  government  withdraws  from  the  programs  to  which 
it  has  been  committed  for  decades  past. 

Unique  as  the  MBL's  message  is,  therefore,  and  uniquely  valuable  for  our 
culture  as  its  services  to  biology  are,  there  isn't  much  time  to  lose  in  fund  raising: 
as  the  competition  mounts,  so  will  the  negative  effects  of  regional  loyalties;  ar- 
guments about  "elitism;"  the  legitimate  outcry  for  replacement  of  social  services; 
and  the  likely  continuation  of  economic  troubles  stemming  from  high  energy  costs 
and  foreign  industrial  competition. 

Progress  toward  the  establishment  of  a  competent,  permanent  in-house  devel- 
opment capability  was  slow  in  1981,  for  reasons  beyond  the  possibility  of  control. 
All  the  work  of  funding  Phase  I  had  therefore  to  be  done  under  the  existing  ar- 
rangements, with  their  heavy  burdens  upon  the  Director,  his  long-suffering  sec- 
retary, and  the  Laboratory's  external  consultants.  Effective  as  those  arrangements 
may  have  been  in  terms  of  dollars  won,  and  by  comparison  with  the  achievements 
of  other  institutions,  they  have  not,  in  my  opinion,  been  effective  enough. 


REPORT  OF  THE  DIRECTOR  37 

I  am  delighted  to  report,  however,  that  in  1982  the  MBL  appointed  a  highly 
qualified  Director  of  Development,  with  whose  person  and  work  many  readers  of 
this  Report  will  have  become  familiar  by  the  time  of  publication.  We  trust  that 
there  will  be  adequate  time  in  which,  with  the  aid  of  the  new  Development  staff, 
we  can  achieve  the  stated  goals  of  the  Second  Century  Fund  Campaign,  and  perhaps 
go  beyond  them,  before  the  Laboratory's  hundredth  birthday. 

Operations 

1.  Financial 

Controller  Edward  Casey  left  the  MBL  in  the  early  spring  of  1981.  The  As- 
sociate Director,  who  remained  in  office  for  some  eight  months  until,  for  personal 
and  professional  reasons,  he  found  it  necessary  to  resign,  undertook  as  part  of  his 
assignment  to  stand  in  for  the  Controller.  That  effort  had  but  limited  success.  The 
Associate  Directorship — a  position  we  have  decided  not  to  fill  again — made  too 
many  other  demands  upon  the  incumbent's  time.  The  search  for  a  successor  to  Mr. 
Casey  did  not  therefore  begin  seriously  for  a  good  many  months. 

Again,  it  gives  me  honest  pleasure  to  report  that  a  critical  management  problem 
was  solved  in  due  course:  in  April  of  1982,  Mr.  John  W.  Speer,  former  chief 
financial  officer  of  Rhode  Island  College,  joined  the  Laboratory  as  its  Controller. 
The  high  expectations  of  his  performance  we  had,  upon  the  basis  of  credentials 
and  his  important  prior  achievements,  are  being  fulfilled.  Objective  evidence  thereof 
is  already  to  be  seen  everywhere  in  the  Laboratory's  financial  activities. 

It  is,  nevertheless,  important  to  note  that  the  Controller's  department,  every 
member  of  it,  had  to  carry  on  for  nearly  a  year  without  the  authority  and  technical 
leadership  of  a  Head.  They  responded  as  MBL  staff  seem  nearly  always  to  have 
done;  quietly,  and  without  complaint.  The  routine  and  the  extra  jobs  were  done: 
accounts  payable,  accounts  receivable,  payroll,  personnel,  the  technically  and  psy- 
chologically demanding  management  of  grants  and  contracts.  They  were  done  well. 

I  would  like  to  think  that  these  colleagues  have  understood  the  sincerity  of  my 
personal  thanks,  and  I  hope  that  somehow  those  of  the  entire  MBL  community 
will  also  be  conveyed  to  them.  I,  for  one,  judge  it  no  slight  achievement  for  the 
Laboratory  to  have  ended  so  complicated  and  management-deficient  a  year  with 
its  finances,  for  all  practical  purposes,  in  balance. 

Among  the  threats  and  bad  auguries  of  the  past  year  in  the  domain  of  federal 
support  for  basic  research,  a  positive  event  stands  out  for  the  MBL  and  the  other 
U.  S.  A.  marine  laboratories.  In  the  early  spring  of  1981,  a  group  of  Directors  of 
those  laboratories  issued  a  brief  report  to  the  National  Science  Foundation.  This 
document*  resulted  from  earlier  meetings,  at  first  of  the  entire  group,  and  later 
of  a  Steering  Committee  elected  to  prepare  the  draft.  Its  central  concern  was  the 
financial  plight  of  marine  laboratories,  especially  those  committed  solely  or  largely 
to  the  study  and  research  utilization  of  marine  plants  and  animals. 

Among  its  recommendations  was  the  establishment  within  the  N.S.F.  of  a  spe- 
cial, inter-program  funding  mechanism,  the  purpose  of  which  would  be  to  provide 
"core"  support  for  the  maintenance  and  improvement  of  capital  facilities  at  these 
laboratories.  To  the  great  credit  of  the  Foundation's  officers,  they  had  provided 

*  U.  S.  Marine  Laboratories:  A  Plan  for  Modernization  and  Maintenance.  J.  D.  Costlow,  P.  R. 
Gross,  C.  Pittendrigh,  R.  R.  Strathmann,  Steering  Committee.  Submitted  to  the  National  Science 
Foundation,  dated  February,  1981,  under  PCM-80- 17003. 


38  MARINE  BIOLOGICAL  LABORATORY 

partial  financial  support  for  the  study,  and  among  them  are  several  who  had  an- 
ticipated its  outcomes. 

As  it  emerged,  there  was  very  little  criticism  of  existing  practices,  but  a  good 
deal  of  argument  for  the  need  to  rectify,  by  new  practices,  a  potential  threat  to  a 
group  of  indispensable  national  research  facilities.  After  study  of  this  report  at  all 
administrative  levels,  the  Foundation  responded  by  announcing  a  new  program, 
quite  close  in  design  to  what  had  been  recommended.  Initial-year  funding  is  much 
less  than  the  amount  suggested,  but  more  than  we  might  have  hoped  for,  considering 
the  Foundation's  already  perilous  budget  situation  for  1982. 

What  is  important  about  these  events,  and  about  the  evidence  of  good  will  and 
understanding  within  the  N.S.F.  administration,  is  not  the  detail  of  first-year  fund- 
ing, nor  the  announced  rules  (which  are  in  fact  reasonable).  Rather,  the  new  pro- 
gram formalizes  recognition,  by  the  key  science  agency  of  government,  that  marine 
biological  laboratories  are  a  national  responsibility;  they  should,  and  probably  will 
some  day  be,  supported  via  more  appropriate  funding  mechanisms.  This  is  no  more 
than  has  been  done  for  many  years  in  behalf  of  blue-water  oceanography,  but  it 
is  heartening  to  know  that  similar  recognition  of  marine  biology  laboratories,  of 
their  missions  and  their  accomplishments  transcending  descriptive  marine  biology, 
has  emerged. 

It  would  be  difficult  to  overstate  the  potential  importance  of  this  for  eventual 
rationalization  of  overhead  cost  recovery  systems  in  institutions  such  as  ours,  quite 
independently  of  the  quantitative  arguments  that  rage,  and  will  continue  to  rage, 
on  the  subject  of  overhead  costs.  For  the  MBL,  where  under-recovery  of  operating 
costs  from  the  grants  in  whose  behalf  those  costs  are  incurred  has  been  a  depressing 
fact  of  life,  this  is  all  good  news. 

2.  Library 

I  shall  not  report  here  upon  the  Library's  record  of  operations  for  the  past  year, 
since  our  Librarian  has  space  of  her  own  for  the  purpose  elsewhere  in  this  volume, 
but  I  cannot  resist  quoting  from  one  of  her  recent  memoranda:  "At  the  present 
moment  two  electricians  are  working  over  my  head  installing  new  lighting,  car- 
penters are  drilling  in  the  hall,  and  tile  men  are  crawling  on  the  future  floor  across 
the  hall.  So — being  unable  to  do  much  of  anything  else  I  shall  sit  in  the  midst  of 
chaos  and  send  you  thoughts.  .  .  ." 

Ms.  Fessenden's  written  thoughts  in  this  form  can  be  pithy,  often  sufficiently 
pithier  than  the  example  to  preclude  their  publishability  in  the  learned  press;  but 
they  always  represent  accurately  the  state  of  mind  of  the  Library  staff.  In  the 
present  instance,  that  state  of  mind  is  influenced  by  Chaos,  but — as  I  replied  to 
her  eventually — What  a  Lovely  Chaos!  After  a  very  few  months  of  it,  we  shall  have 
that  expanded  and  improved  Library  facility  for  which  the  MBL  Corporation,  and 
indeed  the  entire  Woods  Hole  scientific  community,  have  been  agitating  for  decades. 

3.  Buildings  and  Grounds 

Mr.  Robert  Gunning,  who  was  eligible  for  a  well-deserved  and  productive  re- 
tirement in  1982,  has  been  convinced,  against  his  quite  sensible  initial  decision,  to 
remain  on  active  duty  as  Head  of  the  Department  for  another  year.  It  was  a 
generous  decision  under  the  circumstances;  it  means  that  we  shall  have  the  benefit 
of  continuity  in  management  and  technical  direction  of  that  all-important  com- 
ponent of  the  MBL  staff  during  the  rehabilitation  of  the  Lillie  Laboratory. 


REPORT  OF  THE  DIRECTOR  39 

The  negotiations  alluded  to  had  no  negative  effect  upon  the  steady  routine  of 
the  Department.  In  1 98 1 ,  45  laboratories  in  Lillie  were  renovated,  including  removal 
of  the  last  of  Dr.  Drew's  marvelously  impregnable,  obtrusive  concrete  sinks  and 
tables,  and  their  replacement  with  equipment  made  of  stainless  steel  and  fiberglass. 
All  the  old,  cracked  cast  iron  plumbing  has  been  removed  and  replaced  with  fi- 
berglass floor  drains  and  polypropylene  piping;  sea  water  supply  is  now  that  long- 
sought  dual  system.  First  major  steps  in  modernizing  Lillie's  heating  systems  were 
taken,  including  installation  of  thermostatic  steam  radiator  valves  and  remote,  wall- 
mounted  thermostats  in  all  laboratories. 

For  those  many  Corporation  members  and  other  investigators  who  are  concerned 
about  housing,  it  is  pleasant  to  report  that  the  B&G  staff  have  refurbished  com- 
pletely the  dormitory  wing  of  the  Brick  Apartment  House — an  area  that  comprises 
three  apartments,  eighteen  bedrooms,  and  three  bathrooms.  It  will  be  ready  for 
occupancy  in  the  1982  summer  session. 

4.  Marine  Resources 

Having  written  a  great  many  words  of  praise  on  this  subject  in  last  year's 
Director's  Report,  and  since  then  in  several  other  documents,  I  feel  sure  that  more 
of  them  would  be  redundant  here.  Suffice  it  to  say  that  having  the  new  R/V 
GEMMA  at  its  disposal  has  made  the  work  of  the  department  much  easier  than 
in  the  past.  Not  content  with  that  relaxation  of  their  difficulties,  John  Valois  and 
his  staff  have  responded  by  extending — without  having  been  asked — its  benefits  to 
the  entire  MBL  community,  e.g.,  by  adding  another  day  to  the  squid  delivery 
schedule.  Satisfactory  as  GEMMA  has  proven,  there  remains  an  urgent  need  for 
properly  operating  and  well-adapted  vessels.  While  it  was  an  earlier  plan  to  elim- 
inate from  the  fleet  all  wooden  boats,  the  petrochemical  origin  of  polyester  resins 
has  made  the  cost  of  new  fiberglass  boats  prohibitive.  Accordingly,  the  wooden  R/ 
V  CIONA  has  had  a  complete  refit,  including  much  work  on  the  hull  and  deck  rig, 
with  the  result  that  this  familiar  workhorse,  which  has  served  the  MBL  for  twenty 
years,  should  now  have  at  least  another  ten  in  her. 

Not  mentioned  in  earlier  reports  is  the  work  of  the  SCUBA  diving  team  attached 
to  this  Department.  It  consists  of  three  competent  divers,  whose  equipment  and 
work  schedules  are  handled  with  far  greater  care  than  even  the  applicable  regu- 
lations require.  In  consequence,  such  MBL  regulars  among  marine  animals  as 
Microciona,  Spisula,  Chaetopterus,  Metridium,  and  Asterias  continue  to  be  avail- 
able for  research.  If  these  forms  had  to  be  dredged,  rather  than  collected  by  divers, 
they  would  no  longer  be  "available"  in  the  sense  of  utility. 

The  new,  but  already  productive  collaboration  between  the  Marine  Resources 
Department  and  the  Laboratory  for  Marine  Animal  Health  deserves  special  men- 
tion, but  more  appropriately  below,  under  the  head  of  Research  Programs. 

5.  Public  Information 

This  Department  was  known  until  recently  as  "Public  Relations,"  but  the  rep- 
resentations of  an  honored  Trustee,  more  sensitive  to  language  and  titular  affect 
(because  of  his  profession,  as  well  as  in  consequence  of  his  good  literary  genes) 
than  the  rest  of  us,  convinced  us  to  change  the  name.  Barbara  Haskell  was  in 
charge  through  1981  and  until  March,  1982,  at  which  time  she  resigned  because 
of  the  need  to  move  away  from  the  Cape.  This  will  be  a  loss  deeply  felt,  because 
under  Haskell's  direction  and  with  her  painstaking  attention  to  style  and  content, 


40  MARINE  BIOLOGICAL  LABORATORY 

all  the  MBL  publications  coming  from  the  Department  showed  a  discontinuously 
upward  change  in  quality. 

Those  publications  include  more  than  the  familiar  MBL  NEWSLETTER  and 
NEXUS.  Issued  from  the  Department's  office  are  such  ad  hoc  literary  objects  (and 
they  have,  some  of  them,  been  literary)  as  news  releases  to  the  press  and  biograph- 
ical sketches  of  speakers — as  for  the  Friday  Evening  Lectures. 

In  1981  the  Department  undertook  a  number  of  important,  new,  non-recurring 
responsibilities,  such  a;;  collaboration  with  producers  and  other  powerful  persons 
of  the  television  world,  and  with  the  editors  and  staff  writers  of  commercial  and 
corporate  magazines.  These  collaborations  yielded  several  important  television 
events  (e.g.,  segments  of  a  NOVA  program),  a  splendid  article  in  the  Polaroid 
Corporation's  CLOSE-UP,  and  references  to  MBL  science  and  scientists  in  such 
national  publications  as  NEWSWEEK. 

Barbara  Haskell's  able  and  energetic  assistant,  Lee  Anne  Campbell,  has  agreed 
to  serve,  and  is  serving  competently  at  the  time  of  writing,  as  acting  Public  Infor- 
mation Officer,  until  such  time  after  establishment  of  our  new  Development  office 
as  all  concerned  may  consult  together  on  possible  changes  in  organization  and  size 
of  the  Department.  It  is  a  hopeful  sign  for  the  future  that  this,  and  a  few  other 
Departments  at  the  MBL,  have  acquired  the  depth  of  skilled  manpower  to  allow 
such  flexibility. 

6.  The  George  M.  Gray  Museum 

The  principal  function  of  our  Gray  Museum  is  (1)  to  assist  MBL  and  outside 
investigators  and  students  in  the  identification  of  local  and  regional  species  of  plants 
and  animals,  and  (2)  to  supply  otherwise  poorly  accessible  information  on  collection 
and  maintenance  of  organisms.  The  museum  contains  several  thousand  preserved 
specimens  of  local  animals,  as  well  as  sample  forms  inhabiting  the  waters  from 
Maine  to  Virginia.  The  herbarium  contains  about  5,000  sheets,  principally  of  Cape 
Cod  and  Islands  species.  Holdings  of  the  museum  have  been  extensively  catalogued 
and  checklisted:  the  Curators,  of  whom  Dr.  Wesley  N.  Tiffney  is  the  Chief,  are 
available  to  assist  investigators  Monday  through  Friday,  for  a  total  of  20  hours 
per  week.  Their  guidance  is  easily  arranged  for,  as  is  opening  of  the  museum  at 
special  times,  by  appointment.  This  modest,  but  excellently  operated  activity  serves 
several  hundred  users  per  year,  for  each  of  whom  those  services  are  very  important 
indeed. 

7.  Instructional  Programs 

In  1981  the  Laboratory  offered  seven  regular  summer  courses,  each  of  which 
was,  by  the  test  of  critical  external  opinion  (as  mine  is  not:  I  think  that  they're  the 
best  biology  courses  in  the  world),  up  to  the  historic  high  standard  of  the  species. 
The  second  offering  of  our  newest  course,  the  Biology  of  Parasitism,  headed  by 
John  David,  was  even  more  exciting  and  better-received  within  the  community— 
if  such  a  thing  is  possible — than  the  first.  Rudy  Raffs  direction  of  the  Embryology 
course  was  efficient  as  before,  and  the  course  content  remained  a  remarkable  ex- 
ample of  eclecticism  surviving  in  the  midst  of  scholarship  and  high  technical  stan- 
dards. Ivan  Valiela  and  John  Teal  co-directed  the  Marine  Ecology  course  for  the 
fifth  year  and  agreed,  upon  request  of  the  Director  and  the  Committee  on  Instruc- 
tion, to  continue  for  a  sixth  while  the  course's  problems  (entirely  in  the  category 
of  financial  support)  and  strengths  (the  existing  syllabus;  the  extraordinary  op- 
portunities of  surrounding  landscapes;  the  absence  of  such  courses  in  any  of  the 
area  universities)  are  assessed  carefully,  and  financial  support  for  the  successor  is 


REPORT  OF  THE  DIRECTOR  41 

sought.  There  will  be,  I  hasten  to  add,  no  problem  in  recruiting  a  new  Instructor- 
in-Chief.  It  is  our  concern,  however,  to  give  him  a  proper  start. 

Harlyn  Halvorson's  second  year  as  head  of  the  new  Microbial  Ecology  course 
was  eminently  successful,  and  his  unusual  skills  (for  a  distinguished  experimental 
scientist)  in  management  and  fund-seeking  have  benefited  the  students  and  the 
MBL  in  a  multitude  of  ways.  In  the  unique  Neural  Systems  and  Behavior  course, 
Ronald  Hoy  was  joined  this  year  by  Eduardo  Macagno  to  form  a  co-directorate. 
For  this  course,  as  in  all  the  others,  we  now  have  objective,  external  peer-group 
evidence  to  support  our  internal  conviction  (always  dangerous  when  left  to  itself) 
of  excellence. 

Joel  Rosenbaum  succeeded  K.  VanHolde,  for  the  1981  Physiology  offering,  as 
Instructor-in-Chief.  This  old  and  distinguished  course,  which  has  one  of  the  longest 
continuous  records  of  training  grant  support,  has  now  undergone  one  of  its  quin- 
quennial changes  of  direction:  neither  abrupt  nor  in  respect  of  intellectual  rigor, 
but  perceptible  nevertheless.  As  might  be  expected,  it  now  has  a  new  commitment 
to  the  study  of  motility,  cytoskeletal  organization,  and  nucleo-cytoplasmic  inter- 
actions at  that  level,  and  it  is  at  the  forefront  of  the  field. 

I  might  note  here  that  being  at  the  forefront  does  not  preclude  cyclicity:  the 
last  time  this  last  emphasis  was  brought  to  the  course  it  was  done  by  Daniel  Mazia, 
aeons  ago,  when  the  writer  sat  in  on  the  lectures  as  an  excuse  for  not  starting  to 
work  on  his  thesis  at  the  crack  of  dawn. 

John  Hildebrand  and  Tom  Reese  directed  the  Neurobiology  Course  for  their 
second  year,  and  that  had  two  noteworthy  outcomes.  First,  and  by  far  the  more 
important  for  the  writer,  who  must  pay  attention  to  serious  things,  was  their  perfect 
adherence  to  budget,  without  any  noticeable  attrition  of  quality.  Second  was  that, 
in  connection  with  applications  for  continued  funding,  they  sought  some  modest 
testimonials  in  the  course's  behalf  from  a  number  of  the  world's  most  eminent 
neuroscientists.  The  outcome,  which  should  have  been  no  surprise,  since  most  of 
those  are  also  alumni  or  associates  of  the  course,  was  nevertheless  gratifying:  With 
no  exception,  those  asked  wrote  letters  to  the  course  directors  asserting — in  sum- 
that  the  MBL  summer  Neurobiology  course  is  the  only  one,  and  therefore  the  best, 
of  its  kind  in  the  world;  that  it  would  be  an  unthinkable  disservice  to  American 
neurobiology  for  its  support  to  be  diminished  and  its  survival  threatened. 

Gerald  Peters  and  Fred  Ausubel  paid  us  an  extended  visit  during  the  summer 
of  1981  for  purposes  of  planning  the  organization  and  funding  of  our  next  major 
offering  in  the  plant  sciences,  a  pilot  version  of  which  is  to  be  mounted  in  1982, 
and  the  definitive  offering  in  1983,  funding  and  the  goodwill  of  the  vegetable  gods 
permitting. 

1981  versions  of  the  January  semester  and  the  Short  Courses  were  in  the  main 
as  described  in  earlier  reports.  The  details  are  given  elsewhere  in  this  volume.  Both 
series  have  been  a  boon  to  the  Laboratory  and  to  the  participants,  as  the  records 
attest.  There  having  been  some  important  events  and  decisions  for  change  in  these 
programs  in  1982,  I  leave  the  subject  for  much  fuller  discussion  next  year. 

The  Macy  Scholars'  Program  and  the  Steps  Toward  Independence  Program 
made  their  accustomed  contributions  in  1981.  The  comment  applied  to  the  January 
semester,  above,  in  respect  of  1982  changes,  applies  here  as  well. 

Dr.  Morton  Maser,  who  is  Assistant  Director  for  Educational  and  Research 
Services,  has  been  a  dedicated  impresario  of  those  performances  by  which  the  needs 
of  the  instructional  program,  as  disparate  as  Admissions  and  advertising,  on  the 
one  hand,  and  service  laboratories  (e.g.,  EM,  hot  lab),  on  the  other,  are  met. 
Assisting  him  as  Admissions  Officer,  Jane  Leighton  has  maintained  civility  and 
decorum  ("kept  the  lid  on,"  as  one  of  our  patois-prone  instructors  defined  it)  in 


42  MARINE  BIOLOGICAL  LABORATORY 

a  busy  office  with  heavy  potential  for  disturbance  of  the  peace.  I  refer,  thereby, 
to  the  habit  of  other  administrations  with  programs  to  which  many  want  access 
and  few  can  gain  it;  and  for  which  funding  and  student  support  is  a  labyrinth,  of 
hiding  the  Admissions  office  and  officers. 

The  MBL  does  not  hide  its  Admissions  office.  Yet  it  has  been  a  quiet,  efficient, 
and  friendly  place.  This  last  leads  me  to  render  thanks  also  to  Joan  Howard,  Grants 
and  Contracts  Officer  and  a  member  of  the  Controller's  Department,  whose  thread- 
ing the  labyrinth  of  training  and  other  grants,  specifically  in  support  of  our  courses, 
has  been  as  skillful  a  performance  as  Jane  Leighton's. 

Research 

\.  Summer 

The  MBL  was  full  again  in  the  summer  of  1981,  and  the  summer  was  again 
full  in  the  other  sense — of  lectures,  symposia,  and  demonstrations  beyond  the  tra- 
ditionally scheduled  ones.  There  were,  for  example,  not  only  the  expected  Friday 
Evening  Lectures,  but  such  special  events  as  a  lecture  by  Adrian  Horridge,  visiting 
from  Australia,  on  the  insect  compound  eye,  and  Lynn  Margulis's  Associates' 
Lecture  entitled  "The  Earliest  Life  on  Earth,"  making  heavy  and  audience-en- 
thralling use  of  the  magic  lantern.  The  Rockefeller  Foundation  and  the  MBL  co- 
sponsored  a  week-long  special  lecture  series  on  Scientific  Information  Systems  and 
Information  Retrieval,  with  such  participants  as  Kenneth  S.  Warren,  Eugene  Gar- 
field,  William  Goffman,  and  Frederick  Mosteller.  These  sessions  were  well-at- 
tended, and,  fitting  to  the  new  partnership  of  the  MBL  and  the  WHOI  in  library 
matters,  were  held  alternately  at  the  two  institutions. 

It  would  be  asking  for  trouble  to  identify  a  specific  subset  of  the  summer's 
many  research  achievements  as  particularly  noteworthy.  I  can  quell  disbelief  of 
that  assertion  by  analogy,  in  a  domain  with  which  all  readers  are  surely  familiar: 
the  baroque  Concerto  Grosso. 

Those  who  organize  ensembles  to  play  these  wonderful  works  know  that  for 
acceptable  results  in  public  performance,  the  orchestra  can  tolerate  no  weak  players. 
All  must  be  at  least  highly  competent;  by  preference,  excellent.  These  concerti  are 
scored  for  two  groups  of  players  (one  small  and  one  larger).  One  consists,  usually, 
of  the  first-desk  violinists  (two),  a  violist,  a  cellist,  perhaps  cembalo.  These  are 
known  as  the  concertino:  they  play  the  interesting  melodic  lines  and  sound  the 
central  harmonies.  The  second  group  provides  back-up  and  is  known,  collectively, 
as  ripieni  (literally  "fillers"). 

Suffice  it  to  say  that  there  is  conflict:  You  cannot  have  a  collection  of  first-class 
musicians  agree  among  themselves  about  who  is  ripieno  and  who  is  concertino.  Nor 
does  drawing  lots  help.  No  sensible  fiddler  will  risk  on  a  coin-toss  having  to  play 
endlessly  repeated  bass  notes.  He  would  rather  fight.  Yet  you  do  need  equally  good 
musicians;  am  you  do  need  ripieni. 

I  divulge,  for  the  curious,  the  best  of  several  solutions  devised  over  the  centuries 
since  Torelli  and  Corelli,  Vivaldi  and  Boccherini  started  all  the  trouble.  It  works 
for  all  but  the  most  polished  professional  groups,  where  the  need  to  earn  a  living 
transcends  pride,  and  the  players  do  as  they  are  told  in  order  to  get  paid. 

The  trick  is  never  to  emphasize  the  identities  of  lead  and  fill  players.  It  is,  if 
at  all  possible,  to  allow  rotation  from  one  part  to  another  (within  voices),  but  to 
contrive  for  your  most  trustworthy  players  to  be  in  the  first  chairs  on  the  night  of 
a  performance,  having  seen  to  it  that  they  practiced  the  parts  well.  It  is  to  avoid 


REPORT  OF  THE  DIRECTOR  43 

sedulously  any  congratulatory  words  about  individual  parts,  but  to  dwell  instead 
upon  the  marvels  of  the  orchestra  as  a  whole. 

It  should  now  be  clear  why  I  prefer  to  deal  with  the  research  accomplishments 
of  summer  investigators,  in  a  full-house  MBL  summer,  as  though  the  whole  pop- 
ulation were  a  sort  of  Virtuosi  di  Woods' ole.  And  that  would  do  little  violence  to 
the  truth.  They  are.  A  glance  at  a  partial  record — the  published  abstracts  of  the 
General  Scientific  Meetings,  in  the  October  issue  of  the  Biological  Bulletin — will 
bear  me  out.  And  note,  please,  that  it  is  a  partial  record  only.  Not  all  of  the 
summer's  results  are  communicated  in  that  form.  The  eventual  full-length  papers 
that  result  from  a  summer's  work  at  the  MBL  appear  in  a  score  of  different  journals, 
in  several  languages,  and  over  the  course  of  the  next  three  or  four  years. 

Any  subset  of  those  papers,  collected  by  conscious  effort,  sustains  the  conclusion 
obtained  from  independent  tests  of  quality,  e.g.:  research  grant  support  in  this 
competitive  time;  academic  positions  and  honors;  the  eventual  destinations  and  jobs 
of  students  and  postdoctoral  fellows  who  do  research  here  with  the  principal  in- 
vestigators. It  is  that  the  summer  research  population  at  the  MBL,  like  that  of  its 
course  faculty,  is  drawn,  not  from  the  middle  of  a  national  achievement  distribution, 
but  very  much  from  its  high  side. 

I  have  been  accused,  once  or  twice,  of  "elitism"  while  presenting  data  in  support 
of  the  above.  If  that  is  the  definition  of  the  word,  then  so  be  it.  But  I  should  point 
out  that  no  rules  of  the  MBL  except  peer-opinion  require  it  to  be  so,  and  that  the 
geographic,  institutional,  disciplinary,  and  socio-economic  origins  of  the  population 
in  question  are  so  extraordinarily  broad  as  to  make  such  a  definition  fatuous. 

2.   Year  Round  Programs 

For  purposes  of  reporting,  the  year  round  programs  are  much  less  of  a  problem. 
The  year  round  research  group  at  the  MBL  being  of  a  size  with  some  pretty  large 
university  Departments,  it  is  possible  at  least  to  mention  enough  samples,  if  not 
all  of  the  programs,  to  yield  an  impression  of  the  spectrum  of  research  interests 
and  accomplishments.  That,  it  turns  out,  is  worth  doing,  for  the  spectrum,  although 
not  the  size  and  the  resulting  interaction,  is  the  same,  summer  and  year  round. 

For  as  long  as  I  can  remember,  the  MBL  has  had  a  few  distinguished  emeritii 
and  senior  faculty  of  other  institutions  in  residence  and  at  work  the  year  round. 
A  splendid  current  example  of  the  phenomenon  is  D.  Eugene  Copeland,  Professor 
emeritus  of  Tulane  University  and  Trustee  emeritus  of  the  MBL.  Gene  Copeland 
first  retired  and  brought  his  productive  research  program  to  Woods  Hole  in  1977. 
Since  then  he  has  continued,  with  grant  support  from  NIH  and  NSF,  his  important 
fine-structure  studies  on  the  teleost  swim  bladder  and  retina. 

The  work  is  of  physiological  significance,  for  the  swim  bladder  wall  can  retain 
gases  (such  as  O2)  against  pressures  as  high  as  300  atm,  while  there  are  mechanisms 
in  the  eye  that  raise  the  local  oxygen  tension  twenty  times  higher  than  would  be 
produced  by  the  dissociation  of  oxyhemoglobin. 

A  part  of  Copeland's  program  requires  work  on  the  fish  as  soon  as  they  are 
brought  to  the  surface,  and  since  deep-sea  species  are  the  ones  of  interest,  this 
means  work  on  large  oceanographic  vessels.  Dr.  Copeland  has  the  interesting  dis- 
tinction of  being  the  first  MBL  principal  investigator  to  serve  as  a  chief  scientist 
aboard  a  WHOI  research  vessel. 

The  NIH  Laboratory  of  Biophysics,  William  J.  Adelman,  Chief,  is  a  large, 
year  round  contract  program,  i.e.,  one  in  which  the  research  is  done,  effectively, 
"on  location"  with  respect  to  the  parent  organization,  the  National  Institute  of 


44  MARINE  BIOLOGICAL  LABORATORY 

Neurological  and  Communicative  Diseases  and  Stroke.  By  that  mechanism,  the 
lead  scientists  are  employees  of  the  Institute,  while  the  remainder  of  the  program- 
space,  facilities,  staff  (including  scientists) — is  provided  and  managed  by  the  con- 
tractor, the  MBL.  The  Laboratory  of  Biophysics,  Woods  Hole  Unit,  has  two  sec- 
tions, one  on  neural  membranes,  Dr.  Adelman  in  charge,  and  one  on  neural  systems, 
Dr.  Dan  Alkon  in  charge.  Both  these  programs  are  large  enough  to  preclude  even 
a  summary  of  current  activity,  but  activity  there  is  in  good  supply. 

The  section  on  neural  membranes  is  concerned  with  the  structure  and  functions 
of  neural  cells  at  the  ultrastructure  and  molecular  levels.  In  it,  advanced  electron- 
optical,  electrophysiological,  computer,  and  mathematical  methods  are  employed 
in  the  analysis  of  membrane  ionic  channels,  models  for  their  physical  and  electrical 
behavior,  and  the  periodic  structures  of  subcellular  macromolecular  arrays  of  the 
neuroplasm.  Squid  giant  axons  are  the  experimental  material  of  most  common  use, 
but  other  marine  animal  preparations  are  employed  as  well. 

The  section  on  neural  systems  investigates  the  processing  of  information,  espe- 
cially in  reference  to  learning,  in  simple  neural  networks  and  in  the  component 
cells  of  those  networks.  The  preparation  of  primary  interest  in  this  group  is  the 
nudibranch  mollusc,  Hermissenda  crassicornis,  cultured  in  the  laboratory.  A  broad 
range  of  electrophysiological,  biochemical,  morphological,  developmental,  and 
behavioral  experiments  is  carried  out  on  conditioned  animals  and  their  nervous 
systems. 

Among  the  recent  successes  of  this  multilevel  approach  to  a  defined,  whole- 
animal  neural  system  has  been  the  identification  of  complete  sensory  pathways 
responding  to  natural  stimuli  such  as  light  and  gravitational  field.  Changes  in 
associative  learning  behavior  can  now  be  related  specifically  to  altered  properties 
of  individual  motor  neurons. 

This  approach  to  the  cellular  analysis  of  learning,  which  is  simultaneously  in- 
tegrative  and  reductionist,  is  receiving  close  attention  from  cognitive  scientists,  as 
well  as  from  neurobiologists,  across  the  country. 

Dr.  Shinya  Inoue,  who  must  here  represent  a  considerably  larger  group  of 
principal  investigators  in  cell  and  developmental  biology  concerned  with  cell  mo- 
tility  and  morphogenesis,  continues  the  development  of  his  uniquely  sophisticated 
video  microscopy  system.  With  its  aid,  he  and  Dr.  L.  G.  Tilney  have  recently 
visualized  directly,  and  analyzed  the  diffusion-limited  kinetics  of,  actin  polymer- 
ization at  the  tip  of  the  growing  acrosomal  process  (perforatorium). 

Yuchiro  Tanaka,  one  of  the  first  two  recipients  of  a  Jean  and  Katsuma  Dan 
Fellowship,  came  from  Sugashima  to  work  with  Inoue  for  six  months  in  1981.  Dr. 
Tanaka  has  discovered  a  reversible  relaxation  of  the  cleavage  furrow  in  Arbacia 
eggs  treated  with  Cytochalasin  B  or  D.  By  a  combination  of  time-lapse  video 
microscopy  and  tracking  of  cortical  pigment  granules,  local  changes  in  structure 
of  the  cell  cortex  can  be  visualized,  measured,  and  analyzed.  These  studies  promise 
to  contribute  importantly  to  elucidation  of  the  role  of  actin  filaments  in  cytokinesis. 

J.  R.  Whittaker,  one  of  a  group  of  MBL  developmental  biologists  concerned 
with  localization  and  chemical  identification  of  cytoplasmic  morphogenetic  deter- 
minants in  the  early  embryo,  is  also  the  Director  of  the  Boston  University  Marine 
Program  at  the  MBL,  whose  faculty  are  engaged  in  a  broad  range  of  other  dis- 
ciplines as  well  (e.g.,  behavior,  neurophysiology,  cell  biology,  ecology),  and  in  grad- 
uate education.  Whittaker  has  recently  succeeded  in  the  remarkable  feat  of  trans- 
ferring cytoplasm  from  cells  of  the  muscle  lineage  in  ascidian  embryos  to  those  of 
the  epidermal  lineage,  causing  thereby  the  eventual  expression  of  a  characteristic 
muscle  enzymatic  activity  (acetylcholinesterase)  in  progeny  of  the  epidermal  lin- 


REPORT  OF  THE  DIRECTOR  45 

cage,  where  the  activity  would  not  otherwise  appear.  This  opens  the  way  toward 
the  long-sought  test  for  the  chemical  identity  of  a  specific  morphogenetic  deter- 
minant in  a  classically  mosaic  embryo. 

Another  large  year  round  program,  indeed,  the  largest  now  established  at  the 
MBL,  is  the  Ecosystems  Center,  George  M.  Woodwell,  Director.  Its  committed 
grant  support  in  1981  (some  of  it  applicable,  of  course,  to  subsequent  years)  was 
more  than  $5  million.  The  scientific  staff  of  the  Center  are  a  quintessentially  col- 
laborative group,  each  investigator  lending  his  expertise  to  a  range  of  Center  pro- 
jects. The  senior  staff  of  the  Center  includes  a  number  of  internationally  recognized 
figures  in  ecology. 

Biogeochemical  cycles  are  among  the  major  interests  and  their  investigation 
entrains  contributions  from  most  of  the  staff.  In  this  connection,  they  have  recently 
refined  their  estimate  of  the  global  release  of  CO2  to  the  atmosphere  attributable 
to  deforestation.  It  falls  in  the  range  of  2-5  X  109  metric  tons  annually,  which 
figure  is  to  be  compared  with  an  estimated  release  from  combustion  of  fossil  fuels, 
world- wide,  at  5.2  X  109  metric  tons.  The  implication  of  such  figures,  if  both  are 
nearly  correct,  for  the  origins  and  control  of  an  ominously  rising  CO2  load  in  the 
earth's  gaseous  envelope,  will  be  obvious. 

Another  important  contribution  comes  from  the  staff  members  investigating 
sulfur  cycling.  They  have  found  that  a  major  fraction  of  the  net  primary  production 
of  salt  marshes  flows  through  the  sulfur  cycle  of  water  and  sediments  in  the  marsh. 
That  the  complex  transformation  of  sulfur  in  salt  marshes  and  in  other  parts  of 
the  coastal  zone  are  energetically  linked  to  photosynthesis  is  a  significant  finding 
for  the  important  analysis  of  those  transformations. 

The  Laboratory  of  Sensory  Physiology,  Edward  F.  MacNichol,  Director,  and 
Alan  Fein,  Deputy  Director,  accommodates  the  research  of  a  group  of  some  twelve 
resident  investigators  and  up  to  seven  visiting  or  collaborating  scientists.  The  Lab- 
oratory centers  its  investigations  on  the  physiology  and  biophysics  of  vision,  par- 
ticularly on  the  uniquely  favorable  experimental  preparations  available  from  marine 
animals.  The  study  of  cone  pigments  by  microspectrophotometry  of  single  receptors, 
a  technology  in  which  this  laboratory  has  long  been  at  the  forefront,  has  recently 
been  featured  in  major  articles  for  the  general  reader  interested  in  science  (Scientific 
American  and  The  Sciences). 

Other  work  in  progress  and  of  great  importance  in  visual  physiology  deals  with 
the  state  of  Ca,  most  of  which  seems  not  to  be  the  free  ion,  in  the  receptors. 
Identification  of  the  ligand  and  determination  of  its  chemical  structure  is  the  goal 
of  this  work.  A  rapidly  responding  and  reliable  electrode  for  measurement  of  in- 
tracellular  Ca  in  the  ventral  photoreceptors  of  Limulus  has  been  developed,  and 
is  being  employed  for  measurement  of  Ca  during  illumination  and  light  adaptation. 
Results  obtained  thus  far  show  that  while  the  amount  of  intracellular  calcium  does 
change  during  excitation  and  in  the  course  of  adaptation,  the  Ca  concentration  is 
not  a  direct  indicator  of  receptor  sensitivity. 

I  mention  now,  to  complete  this  survey  of  year  round  research,  two  examples 
of  programs  in  marine  biomedicine,  the  only  area  in  which  year  round  activity  at 
the  MBL  has  recently  been  allowed  a  significant  net  increase  in  size,  space,  and 
facilities. 

Dr.  Carol  Reinisch,  Associate  Professor  in  the  Tufts  University  School  of  Vet- 
erinary Medicine,  is  in  residence  at  the  MBL  as  a  condition  of  her  academic 
appointment  and  responsibilities.  The  appointment  represents  a  step  toward  the 
establishment  of  closer,  and  eventually  curricular,  ties  between  that  institution  and 
the  MBL.  Carol  Reinisch's  research  interests  are  in  cellular  immunology  and  in 


46  MARINE  BIOLOGICAL  LABORATORY 

the  pathology  of  marine  invertebrate  animals  (which  subject  she  and  Mrs.  Frederik 
Bang  profess  in  the  MBL's  January  Course  under  that  name). 

An  interesting  example  of  the  confluence  of  her  two  research  interests,  and 
likewise  of  basic  with  applied  research,  is  her  current  work  on  neoplasia  in  Mya 
arenaria.  Aside  from  their  inherent  oncological  interest,  these  studies,  which  have 
been  in  progress  since  October  of  1981,  are  of  practical  toxicological  and  public 
health  value:  the  animals  in  which  tumor  incidence  is  studied  are  collected  (with 
cooperation  of  the  Commonwealth  of  Massachusetts)  from  tidal  flats  closed  to 
shellfishing  because  of  pollution. 

Hematopoietic  tumors  in  these  animals  are  not  rare:  the  incidence  is  in  the 
neighborhood  of  15%.  Dr.  Reinisch  has  generated  a  series  of  monoclonal  antibodies 
(in  Balb/c  By  mice)  to  the  neoplastic  Mya  cells.  At  least  nine  of  these  react  with 
surface  antigens  of  the  tumor  cells,  but  not  with  those  of  normal  cells.  The  work 
has,  therefore,  two  distinct,  implicit  future  directions:  ( 1 )  careful,  large-scale  ep- 
idemiological  studies  of  tumor  incidence,  employing  the  sensitive  new  diagnostic 
tools,  in  "clean"  as  well  as  polluted  environments  (and  using,  also,  Mytilus  for  a 
test  of  the  generality  of  the  Mya  findings);  and  (2)  identification,  using  electro- 
phoresis,  of  the  antigens  being  recognized  by  the  monoclonal  antibodies,  and  their 
subsequent  isolation  and  purification. 

The  Laboratory  for  Marine  Animal  Health,  whose  director  is  Prof.  Louis  Lei- 
bovitz,  of  Cornell  University,  represents  a  new  program  that  is  year  round  offspring 
to  the  flourishing  Aquatic  and  Veterinary  Medicine  Program  ("Aquavet"),  a  col- 
laboration among  the  MBL,  Cornell,  and  the  University  of  Pennsylvania;  and  an 
important  step  toward  equipping  the  entire  marine  resources  effort  at  the  MBL, 
including  the  Department  of  that  name,  for  the  next  decade. 

Such  a  program  of  preparedness,  for  the  one  Department  without  which  research 
at  the  MBL  could  not  proceed  for  more  than  a  few  days,  entails  a  measured  response 
to,  and  a  plan  for  dealing  with,  several  threats:  (1)  instability,  unpredictability,  or 
even  disappearance  of  populations  of  wild  animals  needed  for  researcch  (e.g., 
squid!);  (2)  unidentified,  and  hence  uncontrollable  disease  within  populations  of 
specimens  already  collected  and  held;  (3)  failure  of  the  very  old  wooden  buildings, 
relics  of  simpler  and  easier  times,  in  which  the  high-intensity  and  contemporaneous 
marine  resources  activities  are  housed.  The  list  can  go  on. 

The  third  of  those  threats  has,  as  its  response,  our  plans  for  a  new  Marine 
Resources  Center.  Other  than  to  identify  and  create  programs  of  research  that  will 
share  such  a  facility  with  the  Department  and  its  staff,  there  is  little  more  to  do 
than  to  find  the  money.  The  first  two,  however,  imply  a  newly  urgent  need  for 
whole  volumes  of  scientific  information  that  does  not  yet  exist,  e.g.,  (1 )  the  practical 
ecology  of  forms  needed  now  and  in  the  future  for  research,  and  (2)  a  diagnostic 
pathology  and  clinical  medicine  of  those  animals. 

The  Aquavet  program  is  concerned  with  establishment  and  codification — i.e., 
with  the  creation  of  such  a  discipline.  It  is  a  young  venture,  but  it  has  already 
proven  successful.  It  is  clear  that  the  informed  clinical  approach,  for  which  veter- 
inarians are  trained,  can  and  does  allow  insight  into  the  diseases  of  hitherto  un- 
studied species,  and  that  such  insights  suggest  and  imply  practical  methods  of 
control. 

Dr.  Leibovitz,  who  is  a  distinguished  pathologist,  and  whose  research  activities 
in  that  field  continue  as  before,  except  that  he  is  now  in  residence  at  the  MBL 
year  round,  has  also  established  a  strong  working  relationship  with  John  Valois, 
head  of  the  Marine  Resources  Department,  and  with  its  staff.  Not  unexpectedly 


REPORT  OF  THE  DIRECTOR  47 

for  those  of  us  who  know  Valois,  cooperation  and  interest  have  been  mutual:  the 
Department  has  collaborated  fully  with  the  new  Laboratory  for  Marine  Animal 
Health  (LMAH)  in  mounting  those  of  its  programs  that  require  access  to  Marine 
Resources  facilities  and  procedures.  There  are  already  several  such  programs,  and 
I  can  hope  to  accomplish  no  more  here  than  to  convey,  by  means  of  a  list,  an 
impression  of  their  scope,  their  purposes,  and  their  research  components: 

Morning  Rounds,  for  health  inspection  of  all  animals  maintained  here,  with  a 
new  system  of  records  that  document  numbers  of  animals  held,  numbers  removed, 
morbidity,  and  mortality  each  day. 

Direct  and  immediate  examination  of  sick  and  dead  animals,  followed  by  nec- 
ropsy in  a  special  area  set  aside  for  it,  and,  for  cases  of  interest,  by  detailed 
diagnostic  pathology. 

Regular  water  quality  testing. 

After  a  year  of  operation,  the  LMAH  emerges  as  a  practical  and  valuable 
activity,  certain  to  have  its  descendant(s)  in  a  key  role  in  the  MBL's  planned 
Marine  Resources  Center.  Specific  priorities  for  maintaining  animal  health  have 
already  appeared:  temperature  control,  sediment  control,  prevention  or  minimi- 
zation of  abrasive  injuries.  A  new  program  of  preventive  care  is  in  the  making.  A 
large  number  of  specific  infections  and  contagious  bacterial,  viral,  mycotic,  and 
parasitic  diseases  have  been  diagnosed  in  species  that  are  of  concern  to  us. 

As  the  experimental  facilities  improve,  it  will  be  possible  to  reproduce  these 
diseases  and  to  define  the  specific  pathogenicities.  Therewith,  by  the  classical  se- 
quence of  scientific  medicine,  will  come  control  measures,  and  in  some  cases,  cures. 
Such  information  is  an  indispensable  adjunct  to  data  on  water  quality  limits,  nu- 
tritional requirements,  and  specific  environmental  needs  of  each  species.  It  should 
be  very  much  easier  to  proceed  from  such  a  background  to  mariculture  proper, 
i.e.,  to  raising  animals  of  defined  and  appropriate  genetic  constitution,  entirely  in 
the  laboratory,  than  to  investigate  culture  systems  by  the  "Try  it  and  See  What 
Happens"  method. 

There  is  no  question  that  this  can  be  done:  it  is  already  being  done  here  for  a 
few  species,  and  more  broadly  applicable  biological  (e.g.,  reproductive,  develop- 
mental) and  engineering  data  are  accumulating  rapidly.  What  is  needed  is  to  pre- 
pare deliberately  for  a  time  when  we  shall  need,  or  want,  to  do  the  same  for  any 
other  species  that  we  must  now  hunt  and  capture  in  the  wild. 

H.  B.  Steinbach 

Over  a  long  career  in  teaching  undergraduates,  graduate  students,  and  post- 
doctoral fellows,  I  have  come  to  appreciate  the  wisdom  of  G.  B.  Shaw's  self-analysis: 
he  claimed  to  have  taken  the  greatest  pains  in  deciding  exactly  what  to  say,  and 
then  to  have  said  it  with  utmost  levity.  Although  I  do  not  claim  any  sort  of  kinship 
with  GBS,  it  is  the  method  I  adopted  for  leading  the  reader  into  this  rather  lengthy 
and  serious  Report. 

The  method  fails  consistently,  however,  in  one  kind  of  communication:  that 
having  to  do  with  the  loss  of  a  valued  and  beloved  colleague.  Even  if  the  head  were 
cunning  enough,  the  heart  does  not  permit  it. 

H.  Burr  Steinbach,  former  MBL  Director,  distinguished  scientist,  and  uniquely 
successful  administrator,  died  at  his  home  in  Woods  Hole  on  December  21,  1981. 
All  MBL  regulars  knew  him  well,  for  although  he  was  a  man  who  valued  privacy, 
he  was  also  very  knowable.  This  was,  I  think,  the  result  of  a  great  and  beneficial 
calm  that  lay  at  the  core  of  his  personality. 


48  MARINE  BIOLOGICAL  LABORATORY 

His  mind  was  of  a  complexity  consistent  with  the  pioneering  work  he  did  in 
cellular  physiology,  and  with  his  sensitivity  to  other  complex  people;  yet  he  was 
a  simple  man,  as  easy  for  a  child  to  talk  to  as  for  a  committee  of  Nobel  Laureates. 
He  managed  and  manipulated  some  of  the  most  troublesome  organizations  in  ex- 
istence (university  Biology  Departments  are,  of  course,  high  on  that  list),  and  yet 
he  managed,  somehow,  to  convey  to  everyone  a  perfect  openness. 

For  such  a  man,  usually  cheerful,  able  to  do  whatever  he  wanted  to  do,  unused 
to  any  sort  of  physical  complaint,  it  must  have  been  especially  terrible  to  discover 
himself  in  the  grip  of  an  incurable  neurological  disease,  his  most  valued  physical 
powers,  such  as  that  of  speech,  disappearing  day  by  day.  Yet  even  then  his  simplicity 
and  decency,  his  openness,  remained. 

During  the  final  months,  when  every  few  days  he  would  take  an  increasingly 
difficult  walk  around  Woods  Hole,  he  stopped  regularly  at  our  office  in  Lillie,  and 
conversed  with  us  by  use  of  a  pencil  and  a  writing-pad,  hung  on  him  like  a  necklace. 
Not  a  few  jokes  were  exchanged.  The  other  people  in  the  office,  not  known  for  their 
readiness  to  drop  what  they  are  doing  in  order  to  visit,  did  so  automatically  and 
happily  for  Burr. 

He  died  as  he  had  lived:  with  dignity  and  humanity.  His  is  a  great  loss  that 
we  must  record  among  the  events  of  1981. 


VII.  REPORT  OF  THE  CONTROLLER 

At  the  Executive  Committee  meeting  of  May  8,  1982  during  which  a  consid- 
erable amount  of  time  focused  on  financial  matters,  it  was  suggested  that,  as  a  part 
of  the  annual  financial  report,  the  Controller  highlight  the  more  significant  factors 
that  have  dominated  the  financial  situation  during  the  past  year.  In  response  to 
that  request,  I  am  providing  some  information  that  members  of  the  Corporation 
might  find  useful  in  evaluating  our  financial  performance  during  1981. 

In  unrestricted  Current  Funds,  we  had  a  fund  balance  reduction  of  $43,871 
(exclusive  of  a  $75,087  transfer  to  unrestricted  Plant  Funds).  While  the  financial 
objective  is  a  balanced  budget,  the  small  operating  loss  that  was  experienced  is  not 
considered  significant  in  the  larger  view  of  overall  financial  operations.  This  is 
particularly  so  when  one  realizes  that  the  value  of  MBL  plant  funds  increased  by 
over  $1.5  million,  a  direct  result  of  major  renovation  projects. 

Revenues 

Overall,  unrestricted  revenues  increased  by  almost  5  percent,  which  by  con- 
temporary economic  standards  must  be  considered  at  best  "level  funding."  The  two 
areas  where  reductions  were  experienced  were  in  unrestricted  gifts  and  investment 
income.  Unrestricted  gifts  dropped  by  20  percent,  which  might  be  a  matter  of 
serious  concern  if  it  were  not  for  the  fact  that  our  restricted  gifts,  mostly  from  the 
Candle  House  and  Lillie  renovations,  increased  significantly  over  1980.  In  total, 
gifts  (unrestricted  and  restricted)  amounted  to  over  $1.8  million,  which  was  slightly 
ahead  of  1980. 

Investment  income  was  down  2  percent  from  1980.  This  reflects  a  conscious 
decision  by  the  MBL  to  shift  a  significant  portion  of  its  investments  from  income- 
producing  to  capital-growth  stocks.  While  revenue  was  down  2  percent,  the  value 


REPORT  OF  THE  CONTROLLER  49 

of  the  investment  portfolio  increased  by  5.7  percent,  which  is  encouraging,  given 
the  overall  performance  of  the  stock  market  during  1981. 

Expenditures 

Unrestricted  expenditures  increased  by  17  percent,  reflecting,  for  the  most  part, 
the  continued  and  persistent  problem  of  inflation.  Increases  in  administration  costs 
included  efforts  to  improve  our  development  and  financial  management  programs. 
Instruction  costs  increased  by  39  percent  over  1980,  which  indicates  new  commit- 
ments to  the  educational  programs.  While  "unrestricted"  expenses  for  Library 
support  were  down  1 5  percent,  this  reduction  was  more  than  offset  by  an  increase 
in  "restricted"  funding,  resulting  from  more  effective  use  of  gifts  in  support  of  the 
Library.  In  fact,  aggregate  financial  support  for  the  Library  increased  by  17  percent. 
Costs  of  plant  operations  were  up  by  16  percent,  a  direct  result  of  increases  in 
heating  and  electricity  charges. 

As  we  move  from  1981  to  1982,  the  financial  report  suggests  several  areas  for 
immediate  attention.  First,  efforts  must  be  made  to  improve  support  via  unrestricted 
gifts.  Second,  we  must  continue  attempts  to  increase  overhead  yield.  Third,  a  better 
and  more  timely  system  of  budget  and  financial  control  management  must  be 
implemented.  Finally,  we  must  evaluate  carefully  the  ways  in  which  we  are  spending 
funds  to  ensure  that  expenditures  are  efficient,  effective,  and  optimally  controlled. 


VIII.  REPORT  OF  THE  TREASURER 

The  Laboratory  struggled  to  hold  operating  expenses  within  its  income  in  1981. 
As  the  accompanying  financial  statements  show,  the  effort  was  not  entirely  suc- 
cessful. Gross  income  increased  minimally  in  comparison  with  1980,  but  expenses 
rose  significantly. 

Mindful  of  inflation's  toll,  the  Executive  Committee  has  from  time  to  time 
approved  increases  in  laboratory  space  rents  and  various  use  charges  and  fees. 
Comparison  of  year  to  year  operating  income  items  shows  the  positive  effects  of 
these  actions,  as  well  as  the  caution  with  which  they  have  been  taken. 

Investment  income  again  contributed  importantly  to  gross  income.  In  part,  this 
reflects  the  high  interest  rates  which  continued  through  1981.  Also  significant, 
however,  is  the  fact  that  the  Laboratory's  capital  campaign  generated  funds  which 
were  invested  profitably  while  awaiting  application  to  their  intended  purposes  within 
the  campus  rehabilitation  and  program  plan  (approved  in  1979). 

Inflation  began  to  abate  somewhat,  late  in  1981.  Its  effects  therefore  continued 
through  most  of  the  operating  year  to  pose  difficult  management  problems.  Energy 
costs,  for  example,  again  exceeded  expectations,  causing  plant  operating  expenses 
to  escalate.  Educational  programs  cost  a  good  deal  more  to  mount  in  1981  than 
previously.  Administrative  expenses  also  increased.  The  expenses  of  fund  raising, 
for  example,  were  higher  in  1981  as  the  capital  campaign  gathered  momentum. 
Various  steps  to  provide  needed  depth  to  the  MBL's  management  capability  also 
added  to  the  year's  administrative  costs. 

Stepped-up  efforts  to  deal  with  the  problem  of  delayed  receipts  were  made  in 
1981.  In  most  cases,  the  MBL's  billed  charges  are  paid  by  other  institutions  or  by 
government  agencies.  User  delays  in  approving  and  forwarding  the  Laboratory's 


50  MARINE  BIOLOGICAL  LABORATORY 

invoices,  coupled  with  the  normal  institutional  delays  in  payment  processing,  create 
lags  of  many  months  to  years  in  the  receipt  of  payments.  Fortunately,  the  Labo- 
ratory has  not  yet  had  to  borrow  in  order  to  carry  its  receivables.  Long  payment 
delays  nevertheless  result  in  lost  income,  e.g.,  interest,  on  monies  owed  to  the  MBL. 
Payment  delays  are  especially  unfair  when  the  MBL  finds  itself,  in  effect,  financing 
research  expenses  for  which  funds  are  available  and  reposing  in  other  hands.  For 
this  reason,  the  Executive  Committee  has  endorsed  the  policy  of  requiring  advance 
payment  for  certain  charges,  beginning  and  enforceable  in  1982. 

The  management  and  Executive  Committee  have  given  considerable  attention 
to  those  trends  and  events  which  have  the  potential  of  altering  significantly  the 
Laboratory's  future  ability  to  fulfill  its  missions.  Examples  of  such  critical  concerns 
are  the  effects  of  current  and  likely  future  cutbacks  of  government  sponsorship 
for  research  and  teaching;  the  greatly  increased  competition  for  private  funds;  and 
the  growing  sophistication  of  biological  research  technology,  which  requires  ever- 
greater  investment  in  facilities  and  equipment.  Each  such  issue  contains  a  major 
financial  dimension. 

Faced  with  the  need  to  understand  the  detailed  implications  of  such  trends  and 
events,  the  MBL  is  fortunate  to  have  attracted  to  the  position  of  Controller  a  person 
strongly  qualified  to  direct  the  Laboratory's  financial  administration.  Mr.  John  W. 
Speer,  whose  most  recent  service  was  as  Controller  and  Chief  Financial  Officer  of 
Rhode  Island  College,  joined  the  management  team  of  the  MBL  in  April,  1982. 


REPORT  OF  THE  TREASURER  5  1 


certified  pubtic  accountants 


&Lyrand 


To  the  Trustees  of 

Marine  Biological  Laboratory 

Woods  Hole,  Massachusetts 

We  have  examined  the  balance  sheets  of  Marine  Biological 
Laboratory  as  of  December  31,  1981  and  1980,  and  the  related 
statements  of  current  funds  revenues  and  expenditures  and  changes  in 
fund  balances  for  the  years  then  ended.   Our  examinations  were  made 
in  accordance  with  generally  accepted  auditing  standards  and, 
accordingly,  included  confirmation  from  the  custodians  of  securities 
owned  at  December  31,  1981  and  1980,  and  such  tests  of  the 
accounting  records  and  such  other  auditing  procedures  as  we 
considered  necessary  in  the  circumstances. 

Prior  to  January  1,  1981,  the  Laboratory  capitalized  the 
original  cost  of  land,  buildings  and  related  initial  furnishings  and 
equipment;  while  the  cost  of  subsequent  additions  and  remodeling  was 
expensed  when  incurred.   Generally  accepted  accounting  principles 
require  that  such  additional  additions  and  remodelings  are 
capitalized  and  depreciated  over  their  estimated  useful  lives. 

In  our  opinion,  except  for  the  effect  on  the  1980 
financial  statements  of  the  matter  discussed  in  the  preceding 
paragraph,  the  financial  statements  referred  to  above  present  fairly 
the  financial  position  of  Marine  Biological  Laboratory  at  December 
31,  1981  and  1980,  and  its  current  funds  revenues  and  expenditures 
and  the  changes  in  fund  balances  for  the  years  then  ended,  in 
conformity  with  generally  accepted  accounting  principles  applied  on 
a  consistent  basis,  except  for  the  change,  with  which  we  concur,  in 
the  method  of  accounting  for  capitalization  of  fixed  assets  as 
described  in  Note  C. 


Boston,  Massachusetts 
May  10,  1982 


>/lk^*^«sC 


<? 


52  MARINE  BIOLOGICAL  LABORATORY 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEETS 
December  31,  1981  and  1980 

Assets  1981  1980 

Current  funds: 
Unrestricted: 

Cash  and  savings  deposits                                      $      212,262  $      363,907 

Money  market  securities  1,850,000  1,250,000 
Accounts  receivable,  net  of 

allowance  for 

uncollectible  accounts  623,658  728,611 

Other  assets  19,531  5,004 
Due  from  (to)  restricted 

current  funds  (597,747)  105,104 

Due  to  invested  funds  (90,133)  (26,669) 

Due  to  restricted  plant  fund  (720,535)  (1,052,224) 

Total  unrestricted  1,297.036  1,373,733 

Restricted: 

Accounts  receivable  346,828  733,431 
Investments,  at  cost  (Notes 

B  and  F)  2,179,531  2,085,227 
Due  from  (to)  unrestricted 

current  fund  597,747  (105,104) 

Due  from  invested  funds  350,967  350,967 

Total  restricted  3,475,073  3,064,521 


Total  current  funds  $  4,772,109  $  4,438,254 

Invested  funds: 

Investments,  at  cost  (Notes  B 

and  F)  4,488,885  4,219,999 

Due  from  unrestricted  current 

fund  90,133  26,669 

Due  to  restricted  current  funds  (350,967)  (350,967) 

Total  invested  funds  $  4,228,051  $  3,895,701 

Plant  funds: 

Unrestricted: 

Land,  buildings  and 

equipment  (Note  C)  14,907,184  12,940,384 

Less  accumulated 

depreciation  4.843.425  4.535.825 

Total  unrestricted  10.063.759  8.404.559 

Restricted: 

Due  from  unrestricted 

current  fund  720,535  1,052,224 


Total  restricted  720,535  1,052,224 


Total  plant  funds  $10,784,294  $9,456,783 


The  accompanying  notes  are  an  integral  part  of  the  financial  statements. 


REPORT  OF  THE  TREASURER  53 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEETS 
December  31,  1981  and  1980 

Liabilities  and  Fund  Balances  1981  1980 

Current  funds: 
Unrestricted: 

Accounts  payable  and 

accrued  expenses  $      530,917  $    490,305 

Deferred  income  77,138  75,489 

Fund  balance  688.981  807,939 

Total  unrestricted  1,297,036  1,373,733 

Restricted  funds: 

Unexpended  gifts  and  grants  3,373,696  2,975,128 

Unexpended  income  of 

endowment  funds  1Q1>377  89,393 

Total  restricted  3.475.073  3.064.521 

Total  current  funds  $4,772,109  $4,438,254 


Invested  funds: 

Endowment  funds  2,218,669  2,077,500 

Quasi-endowment  funds  934,143  934,143 

Retirement  fund  (Note  D)  1,075.239  884.058 

Total  invested  funds  $  4,228,051  $3,895,701 


Plant  funds: 

Unrestricted  10,063,759  8,404,559 

Restricted  720.535  1,052,224 

Total  plant  funds  $10,784.294  $9,456.783 

The  accompanying  notes  are  an  integral  part  of  the  financial  statements. 


54 


MARINE  BIOLOGICAL  LABORATORY 


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58  MARINE  BIOLOGICAL  LABORATORY 

MARINE  BIOLOGICAL  LABORATORY 

NOTES  TO  FINANCIAL  STATEMENTS 

A.  Purpose  of  the  Laboratory: 

The  purpose  of  Marine  Biological  Laboratory  (the  "Laboratory")  is  to  establish  and  maintain  a 
laboratory  or  station  for  scientific  study  and  investigations,  and  a  school  for  instruction  in  biology 
and  natural  history. 

B.  Significant  Accounting  Policies: 

Basis  of  Presentation — Fund  Accounting 

In  order  to  ensure  observance  of  limitations  and  restrictions  placed  on  the  use  of  resources  available 
to  the  Laboratory,  the  accounts  of  the  Laboratory  are  maintained  in  accordance  with  the  principles 
of  "fund  accounting."  This  is  the  procedure  by  which  resources  are  classified  into  separate  funds 
in  accordance  with  activities  or  objectives  specified.  In  the  accompanying  financial  statements, 
funds  that  have  similar  characteristics  have  been  combined. 

Externally  restricted  funds  may  only  be  utilized  in  accordance  with  the  purposes  established  by 
the  source  of  such  funds.  However,  the  Laboratory  retains  full  control  over  the  utilization  of 
unrestricted  funds.  Restricted  gifts,  grants,  and  other  restricted  resources  are  accounted  for  in  the 
appropriate  restricted  funds.  Restricted  current  funds  are  reported  as  revenue  when  expended  for 
current  operating  or  other  purposes.  Unrestricted  revenue  is  reported  as  revenue  in  the  unrestricted 
current  fund  when  earned. 

Endowment  funds  are  subject  to  restrictions  requiring  that  the  principal  be  invested  with  income 
available  for  use  by  the  Laboratory.  Quasi-endowment  funds  have  been  established  by  the  Lab- 
oratory for  the  same  purposes  as  endowment  funds;  however,  any  portion  of  these  funds  may  be 
expended. 

The  financial  statements  for  1981  and  1980  reflect  certain  changes  in  format  and  presentation  of 
the  various  funds.  These  changes  have  been  made  by  the  Laboratory  to  distinguish  and  identify 
the  specific  nature  of  certain  restricted  funds.  Other  ^classifications  of  amounts  previously  reported 
have  been  made  to  enhance  the  comparability  of  the  financial  statements. 

Investments 

Investments  purchased  by  the  Laboratory  are  carried  at  cost.  Investments  donated  to  the  Labo- 
ratory are  carried  at  fair  market  value  at  date  received.  For  determination  of  gain  or  loss  upon 
disposal  of  investments,  cost  is  determined  based  on  the  average  cost  method. 

Investment  Income  and  Distribution 

The  Laboratory  follows  the  accrual  basis  of  accounting  except  that  investment  income  is  recorded 
on  a  cash  basis.  The  difference  between  such  basis  and  the  accrual  basis  does  not  have  a  material 
effect  on  the  determination  of  investment  income  earned  on  a  year-to-year  basis. 

Investment  income  includes  income  from  the  investments  of  specific  funds  and  from  the  pooled 
investment  account.  Income  from  the  pooled  investment  account  is  distributed  to  the  participating 
the  basis  of  the  market  value  at  the  beginning  of  the  quarter,  adjusted  for  the  cost  of 
any  additions  or  disposals  during  the  quarter. 


REPORT  OF  THE  TREASURER  59 

C.   Land,  Buildings  and  Equipment: 

Following  is  a  summary  of  the  unrestricted  plant  fund  assets: 

Classification  1981  1 980 

Land  $      719,798  $      639,693 

Buildings  12,535,197  10,694,543 

Equipment  1,652,189  1,606,148 


14,907,184  12,940,384 

Less  accumulated  depreciation  4,843,425  4,535,825 


$10,063,759  $  8,404,559 


The  original  cost  of  land,  buildings  and  related  initial  furnishings  is  capitalized  when  assets  are 
acquired.  Prior  to  January  1,  1981  the  cost  of  subsequent  additions  and  remodeling  was  expensed 
when  incurred  which  amounted  to  approximately  $135,000  in  1980.  Effective  January  1,  1981  the 
Laboratory  adopted  the  accounting  policy  of  capitalizing  such  additions  and  remodeling  in  ac- 
cordance with  generally  accepted  accounting  principles.  For  the  year  ended  December  31,  1981 
this  change  in  accounting  principle  increased  land,  buildings  and  equipment  by  $794,000  and 
depreciation  expense  by  $2,000.  The  financial  statements  have  not  been  restated  for  the  cumulative 
effect  of  this  change  since  the  amounts  are  not  determinable. 

Depreciation  is  computed  using  the  straight-line  method  over  estimated  useful  lives. 

D.  Retirement  Fund: 

The  Laboratory  has  a  noncontributory  pension  plan  for  substantially  all  full-time  employees  which 
complies  with  the  requirements  of  the  Employee  Retirement  Income  Security  Act  of  1974.  The 
actuarially  determined  pension  expenses  charged  to  operations  in  1981  and  1980  were  $137,009 
and  $  1  1 7,557,  respectively.  The  Laboratory's  policy  is  to  fund  pension  costs  accrued,  as  determined 
under  the  aggregate  level  cost  method.  As  of  the  latest  valuation  date,  based  on  benefit  information 
obtained  January  1,  1982,  the  actuarial  present  values  of  vested  and  nonvested  benefits,  assuming 
an  investment  rate  of  return  of  6%,  were  approximately  $955,479  and  $39,561,  respectively.  At 
January  1,  1982  net  assets  of  the  plan  available  for  benefits,  were  approximately  $1,055,861. 

E.  Pledges  and  Grants: 

As  of  December  31,  1981  and  1980,  the  following  amounts  remain  to  be  received  on  gifts  and 
grants  for  specific  research  and  instruction  programs,  and  are  expected  to  be  received  as  follows: 

December  31,  1981  December  31,  1980 


Unrestricted  Restricted  Unrestricted  Restricted 

1981  $104,000  $1,061,356 

1982  $20,000  $  96,800  50,000  66,333 

1983  95,000  5,000 

1984                    40,000                     

$20,000  $231,800  $159,000  $1,127,689 


In  February  1979,  the  Laboratory  initiated  the  MBL  Second  Century  Fund,  a  phased  effort,  to 
secure  $23  million  in  support  of  capital  rehabilitation,  new  construction,  and  endowment.  As  of 
December  31,  1981,  the  Laboratory  has  received  pledges  related  to  this  effort  of  approximately 
$4,000,000  of  which  a  substantial  portion  has  been  collected. 


60 


MARINE  BIOLOGICAL  LABORATORY 


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62  MARINE  BIOLOGICAL  LABORATORY 

IX.  REPORT  OF  THE  LIBRARIAN 

It  was  a  year  of  planning  and  designing  for  the  expansion  of  library  space.  The 
present  collection  is  literally  bursting  off  the  shelves  onto  the  side  counters  and 
tables,  so  the  additional  5,000  square  feet  of  space  will  be  invaluable  over  the  next 
ten  years. 

Discussions  began  in  January  with  architects,  a  library  consultant,  users,  and 
staff.  The  final  pi  ill  include  the  following: 

( 1 )  A  towe    will  be  constructed  at  the  back  of  the  Lillie  Building  to  house  a 

:enter  staircase  and  elevator  and  provide  access  to  all  five  stacks  and  three 
main  floors. 

(2)  Wet  labs  on  the  third  floor  which  are  presently  over  library  space  will  be 

eliminated.  This  area  will  become  the  future  space  for  the  entire  "book" 
collection. 

(3)  The  first  floor  administration  area  will  house  the  archives  and  rare  book 

collection,  future  microform  machines  and  materials,  and  a  conference 
room  for  Lillie  scientists  and  library  users. 

Demolition  and  construction  will  begin  in  January,  1982,  to  be  completed  by 
the  first  of  May  in  order  to  be  ready  for  the  summer  users. 

In  1981  we  added  66  new  journal  titles  to  the  collection,  purchased  2,031  books, 
and  filled  over  4000  inter-library  loan  requests.  We  also  added  a  5600  Xerox 
machine  to  the  copy  service  center,  a  Wang  word  processor,  and  an  electronic 
typewriter  for  cataloging.  Over  600  computer  bibliographic  searches  were  com- 
pleted by  three  staff  members  who  attended  nine  computer-update  training  sessions 
during  the  year. 

X.  EDUCATIONAL  PROGRAMS 

SUMMER 

BIOLOGY  OF  PARASITISM 

Instructor-in-chief 

DAVID,  JOHN,  Harvard  Medical  School 

Other  faculty,  staff,  and  lecturers 

ASKENASE,  PHILIP,  Yale  University  School  of  Medicine 

BANG,  FREDERIK,  Johns  Hopkins  University 

CARTER,  RICHARD,  National  Institutes  of  Health 

CAULFIELD,  JOHN,  Harvard  Medical  School 

CROSS,  GEORGE,  Wellcome  Research  Laboratories,  United  Kingdom 

DAVID,  PETER,  Harvard  Medical  School 

DAVID,  ROBERTA,  Harvard  Medical  School 

DESSEIN,  ALAIN,  Harvard  Medical  School 

FEARON,  DOUGLAS,  Harvard  Medical  School 

GERSHON,  RICHARD,  Yale  University  School  of  Medicine 

GITLER,  CARLOS,  The  Weizmann  Institute  of  Science,  Israel 

HARN,  DONALD,  Harvard  Medical  School 

HOMMEL,  MARCEL,  Harvard  Medical  School 

KAFATOS,  FOTIS,  Harvard  University 

K.ARNOVSKY,  MANFRED,  Harvard  Medical  School 

KELJSCH,  GERALD,  Tufts  University 


EDUCATIONAL  PROGRAMS  63 

MARSDEN,  PHILIP,  Universidade  de  Brasilia,  Brazil 

MAY,  ROBERT,  Princeton  University 

MILLER,  Louis,  National  Institutes  of  Health 

MOSER,  GINA,  Harvard  Medical  School 

NELSON,  GEORGE,  Liverpool  School  of  Tropical  Medicine,  United  Kingdom 

NUSSENZWEIG,  RUTH,  New  York  University  Medical  Center 

PEREIRA,  MIERCIO,  New  England  Medical  Center 

PFEFFERKORN,  ELMER,  Dartmouth  Medical  School 

PIESSENS,  WILLY,  Harvard  Medical  School 

PRATT,  DIANE,  Harvard  Medical  School 

RIFKIN,  MARY,  Rockefeller  University 

ROBERTS,  BRYAN,  Harvard  Medical  School 

SHER,  ALAN,  National  Institutes  of  Health 

SHERMAN,  IRWIN,  University  of  California  at  Riverside 

SIMPSON,  LARRY,  University  of  California  at  Los  Angeles 

SPIELMAN,  ANDREW,  Harvard  School  of  Public  Health 

VINCENT,  ALBERT,  University  of  Southern  Florida  Medical  Center 

WARREN,  KENNETH,  The  Rockefeller  Foundation 

WIRTH,  DYANN,  Harvard  University 

Students* 

*AVILA,  EVA,  Centre  de  Investigacion  y  Estudios  Avanzados,  Mexico 

*DUERR,  ANN,  Massachusetts  Institute  of  Technology 

*LIBERTI,  PIETRO,  Cell  Biology  Laboratory,  Italy 

*LICHTENSTEIN,  LAWRENCE,  Johns  Hopkins  University 

*MENDIS,  KAMINI,  University  of  Sri  Lanka,  Sri  Lanka 

*OCKENHOUSE,  CHRISTIAN,  New  York  University  Medical  Center 

*PERLER,  FRANCINE,  New  England  Biolabs,  Inc. 

*PHILIPP,  MARIO,  National  Institute  for  Medical  Research,  United  Kingdom 

*SAFRANEK,  Louis,  Harvard  University 

*SANTOS,  ISABEL,  Universidade  Federal  do  Rio  de  Janeiro,  Brazil 

*SHAPIRO,  STUART,  International  Laboratory  for  Research  on  Animal  Disease,  Kenya 

*SIDNER,  RICHARD,  University  of  Cincinnati 

*So,  MAGGIE,  Cold  Spring  Harbor  Labs 

*UNBEKANT,  LINDSEY,  Harvard  University 

*WAHLGREN,  CARL,  University  of  Stockholm,  Sweden 

*WINCHELL,  ELLEN,  Johns  Hopkins  University 

EMBRYOLOGY 

Instr  uctor-  in-chief 

RAFF,  RUDOLF,  Indiana  University 

Other  faculty,  staff,  and  lecturers 

BRANDHORST,  BRUCE,  McGill  University,  Canada 
BRUSKIN,  ARTHUR,  Indiana  University 
COLOT,  HILDUR,  Brandeis  University 
DANILCHIK,  MICHAEL,  University  of  Washington 
EDGAR,  ROBERT,  University  of  California  at  Santa  Cruz 
GUERRIER,  PIERRE,  Station  Biologique,  France 
HYMAN,  LINDA,  Brandeis  University 
JEFFERY,  WILLIAM,  University  of  Texas 

'  All  summer  students  listed  completed  the  formal  course  programs.  Asterisk  indicates  those  com- 
pleting post-course  research  sessions. 


64  MARINE  BIOLOGICAL  LABORATORY 

KLEIN,  WILLIAM,  Indi  na  University 

KUSCH,  MEREDITH,  University  of  California  at  Santa  Cruz 

MOON,' RANDALL,  Univ    sity  of  Washington 

MOREAU,  MARC,  Station  Biologique,  France 

NETO,  RODRIGO,  Instituto  de  Biofisica,  Brazil 

RAFF,  BETH,  Indiana  University 

RANKIN,  MARY  ANN,  University  of  Texas 

ROSBASH,  MICHAEL,  Brandeis  University 

RUDERMAN,  JOAN,  Harvard  Medical  School 

SHOWMAN,  RICHARD,  Indiana  University 

SOWERS,  Louis,  Indiana  University 

TYNER,  ANGELICA,  Indiana  University 

VACQUIER,  Vic,  Scripps  Institution  of  Oceanography 

Students1 

*ADELSON,  DAVID,  University  of  Hawaii 
*BALTUCH,  GORDON,  Harvard  College 
*BARNETT,  FAITH,  Harvard  University 

*  BROWN,  ELIZABETH,  University  of  Washington 

*  FERGUSON,  JAMES,  Iowa  State  University 
*GORALSKI,  THOMAS,  Indiana  University 

*GRODEN,  JOANNA,  Cornell  Graduate  School  of  Medical  Sciences 

*  HENDERSON,  JUDITH,  State  University  of  New  York  at  Buffalo 
*HOEFEN,  PAULA,  Pennsylvania  State  University 

*  KEEN  AN,  KATHERINE,  Yeshiva  College 
*KROTOSKI,  DANUTA,  Tulane  University 
*LiN,  PETER,  Johns  Hopkins  University 
*McKiNLEY,  DANA,  University  of  Miami 
*MURTIF,  VICKI,  Yale  University 

'NICOSIA,  ROBERTO,  Medical  College  of  Pennsylvania 
*O'BROCHTA,  DAVID,  University  of  California  at  Irvine 
*OLIVEIRA,  ANA,  Universidade  Federal  Do  Rio  De  Janeiro,  Brazil 
*SHEPARD,  RICHARD,  University  of  Texas  at  Austin 
*SHERMAN,  BETH,  State  University  of  New  York  at  Stony  Brook 
*SKUSE,  GARY,  Syracuse  University 
*SPAIN,  LISA,  Indiana  University 
TUFARO,  FRANK,  McGill  University,  Canada 
*WELLS,  DAN,  Indiana  University 

MARINE  ECOLOGY 

Instructors-in-chief 

TEAL,  JOHN,  Woods  Hole  Oceanographic  Institution 
VALIELA,  IVAN,  Boston  University 

Other  faculty,  staff,  and  lecturers 

ANDERSON,  DON,  Woods  Hole  Oceanographic  Institution 

CARACO,  NINA,  Boston  University 

CONNELL,  JOSEPH,  University  of  California  at  Santa  Barbara 

DACEY,  JOHN,  Woods  Hole  Oceanographic  Institution 

GIBLIN,  ANNE,  Boston  University 

GRASSLE,  FREDERICK,     roods  Hole  Oceanographic  Institution 

HARGRAVE,  BARRY,  Bedford  Institute  of  Oceanography,  Canada 

HOBBIE,  JOHN,  Marine  Biologica     .aboratory 

HORGAN,  ERICH,  Marine  Bio!          i  Laboratory 


EDUCATIONAL  PROGRAMS  65 


HUMES,  ARTHUR,  Boston  University 
JACKSON,  J.,  Johns  Hopkins  University 
JANNASCH,  HOLGER,  Woods  Hole  Oceanographic  Institution 
KOEHL,  MIMI,  University  of  California  at  Berkley 
KORNBERG,  HANS,  Cambridge  University,  United  Kingdom 
LEVINTON,  JEFF,  State  University  of  New  York  at  Stony  Brook 
MADIN,  LARRY,  Woods  Hole  Oceanographic  Institution 
MANN,  ROGER,  Woods  Hole  Oceanographic  Institution 
NIXON,  SCOTT,  University  of  Rhode  Island 
ODUM,  WILLIAM,  University  of  Virginia 
PETERSON,  BRUCE,  Marine  Biological  Laboratory 
PETERSON,  SUSAN,  Woods  Hole  Oceanographic  Institution 
RIETSMA,  CAROL,  State  University  of  New  York  at  New  Paltz 
SEBENS,  KENNETH,  Harvard  University 
WATKINS,  WILLIAM,  Woods  Hole  Oceanographic  Institution 
WILTSE,  WENDY,  Williams  College/Mystic  Seaport 
WITLACH,  ROBERT,  University  of  Connecticut 

Students^ 

BAUER,  JAMES,  Boston  University 

CHOW,  GEORGE,  State  University  of  New  York  at  Buffalo 
*DAVIS,  JONATHAN,  Yale  University 
*HALS,  GARY,  Capital  University 
*HUNTER,  JUDY,  Auburn  University 
*KEEN,  SUSAN,  University  of  Michigan 
*LERER,  DEBRA,  University  of  Massachusetts 
*LIGHT,  JEFFREY,  University  of  Colorado 
*MARKS,  BARBARA,  Johns  Hopkins  University 
*MERCURIO,  KIMBERLEY,  Ohio  Wesleyan  University 

RADCLIFFE,  GEORGE,  Cathedral  High  School 
*SHERRELL,  ROBERT,  Columbia  University 

STEPHAN,  DIANNE,  State  University  of  New  York  at  Syracuse 

STOECKEL,  MARK,  Lafayette  College 

WAGNER,  JEFFREY,  State  University  of  New  York  at  Buffalo 
*WAGNER,  WENDY,  Hanover  College 
*WALCH,  MARIANNE,  Harvard  University 
*WRIGHT,  ANSON,  Harvard  University 

WYNES,  DAVID,  Nichols  College 

MICROBIAL  ECOLOGY 

Instructor-in-chief 

HALVORSON,  HARLYN,  Brandeis  University 

Other  faculty,  staff,  and  lecturers 

ALEXANDER,  MARTIN,  Cornell  University 

ATWOOD,  KIMBEL,  Columbia  University 

CITRI,  NATHAN,  Hebrew  University,  Israel 

COHEN,  YEHUDA,  Hebrew  University,  Israel 

DEMAIN,  ARNOLD,  Massachusetts  Institute  of  Technology 

DWORKIN,  MARTIN,  University  of  Minnesota 

GIBSON,  JANE,  Cornell  University 

GRASSLE,  FREDERICK,  Woods  Hole  Oceanographic  Institution 

GREENBERG,  E.  P.,  Cornell  University 

HASTINGS,  J.  WOODLAND,  Harvard  University 


66  MARINE  BIOLOGICAL  LABORATORY 

HOWE,  BRIAN,  Marine  Biological  Laboratory 

IMHOFF,  JOHANNES,  Institut  Fur  Mikrobiologie,  Germany 

JANNASCH,  HOLGER,  Woods  Hole  Oceanographic  Institution 

KAPLAN,  HEIDI,  Cornell  University 

KEYNAN,  ALEX,  Hebrew  University,  Israel 

KLAR,  AMAR,  Cold  -      mg  Harbor  Labs 

KORNBERG,  HAN"         r abridge  University,  United  Kingdom 

LEADBETTER,  I  /ersity  of  Connecticut 

MACLOEB,  i  LOSEK  i,  MoGill  University,  Canada 

MAHLER,  HENRY,  University  of  Texas 

MARGUIJS,  LYNN,  Boston  University 

PECK,  HARRY,  University  of  Georgia 

POINDEXTER,  JEANNE,  Public  Health  Research  Institute  at  New  York  City 

REZNIKOFF,  WILLIAM,  University  of  Wisconsin 

ROMESSER,  JAMES,  Dupont  Corporation 

ROSENBERG,  EUGENE,  Tel  Aviv  University,  Israel 

ROWND,  ROBERT,  University  of  Wisconsin 

SHAPIRO,  JAMES,  University  of  Chicago 

SHILO,  MOISHE,  Hebrew  University,  Israel 

VALIELA,  IVAN,  Boston  University 

VINCENT,  WALTER,  University  of  Delaware 

Students* 

*BINGHAM,  PETER,  Harvard  College 
*BRILL,  HOWARD,  Cornell  University 
*CASTELFRANCO,  ANN  MARIE,  University  of  Iowa 
*CAVANAUGH,  COLLEEN,  Harvard  University 
*DUROJAIYE,  MUSTAPHA,  Atlanta  University 
*HALL,  VALERIE,  Nantucket  High  School 
*HAMLETT,  NANCY,  Towson  State  University 
*HEUER,  ANN,  California  State  University 

*HONG,  WEK-HEE,  Korea  Advanced  Institute  of  Science  and  Technology,  Korea 
*JURICK,  RICHARD,  University  of  Hawaii 
*KEMP,  CHRISTOPHER,  National  Institutes  of  Health 
*KUDLACZ,  JUDY,  University  of  Nebraska  at  Lincoln 

*MILLER,  MOLLY,  Massachusetts  Institute  of  Technology/Woods  Hole  Oceanographic  In- 
stitution 

*POONAM,  GULATI,  Cornell  University 
*ROBERTS,  SUSAN,  University  of  California  at  San  Diego 

*ROMERO-JARERO,  JORGE,  Centro  De  Ciencias  Del  Mar  Y  Limnologia,  Mexico 
*SLOCK,  JAMES,  Viterbo  College 
*SPENCER,  DAVID,  Indiana  University 
*TOMEI,  FRANCISCO,  Harvard  University 
*VAN  RUN,  JAAP,  Hebrew  University  of  Jerusalem,  Israel 

NEURAL  SYSTEMS  AND  BEHAVIOR 

Instructors-in-chief 

HOY,  RONALD,  Cornell  University 
MACAGNO,  EDUARDO,  Columbia  University 

Other  faculty,  staff,  and  lecturers 

BENNETT,  MICHAEL,  Albert  Einstein  College  of  Medicine 
"ALABRESE,  RON,  Harvard  University 
CAREW,  THOMAS,  Columbia  University 


EDUCATIONAL  PROGRAMS  67 

DeRiEMER,  SUSAN,  Yale  University 

DERBY,  CHARLES,  Boston  University 

EISNER,  THOMAS,  Cornell  University 

HARRIS- WARRICK,  RON,  Cornell  University 

HUBER,  FRANZ,  Max  Planck  Institute,  Germany 

KAHLE,  GUNTHER,  Free  University  of  Berlin,  Germany 

KELLEY,  DARCY,  Princeton  University 

KRONENBERG,  FREDI,  New  York,  NY 

LAYTON,  J.  KIERAN,  Princeton  University 

LEVIN,  MARGARET,  Columbia  University 

LEVINTHAL,  CYRUS,  Columbia  University 

LEVINTHAL,  FRANCOIS,  Columbia  University 

MENZEL,  RANDOLF,  Free  University  of  Berlin,  Germany 

MURPHY,  ROD,  State  University  of  New  York  at  Albany 

NELSON,  MARGARET,  Cornell  University 

NOTTENBOHM,  FERNANDO,  Rockefeller  University 

PEARSON,  KEIR,  University  of  Alberta,  Canada 

THOMAS,  JEFF,  Columbia  University 

WALRATH,  DANA  ELIZABETH,  Columbia  University 

WEEKS,  JANICE,  University  of  Washington 

WOHLERS,  DAVID,  Cornell  University 

Students1 

*BRODFUEHRER,  PETER,  University  of  Virginia 

CARLIN,  NORMAN,  Harvard  University 

DON  CARLOS,  LYDIA,  Northeastern  Ohio  Universities  College  of  Medicine 

DUMONT,  JAMES,  Stanford  University 

ELROD,  SCOTT,  Earlham  College 
*  FALLS,  DOUGLAS,  State  University  of  New  York  at  Albany 

GROSOF,  DAVID,  Harvard  University 

HALLANGER,  ANN,  University  of  Wisconsin  at  Madison 

JUNG,  LADONNA,  Columbia  University 

KAULEN,  PETER,  Free  University  of  Berlin,  Germany 
*KRIEGER,  CHARLES,  McGill  University,  Canada 
*MooRE,  DARRELL,  University  of  Texas  at  Austin 

NOVICKI,  ANDREA,  University  of  Hawaii 

PALLAS,  SARAH,  Cornell  University 

POWELL,  SUSAN,  University  of  Oregon 

QUINN,  RICHARD,  University  of  North  Carolina  at  Charlotte 

RAMIREZ,  RAFAEL,  Institute  Venezolano  de  Investigaciones  Cientificas,  Venezuela 

RAYMOND,  LYNN,  Albert  Einstein  College  of  Medicine 

ROBERTSON,  GAIL,  Washington  University  School  of  Medicine 
*SHAMMA,  SHIHAB,  Stanford  University 

VALINSKI,  WENDY,  West  Virginia  University 

YOUNG,  ROBERT,  Armed  Forces  Radiobiology  Research  Institute 

NEUROBIOLOGY 
Instructors-in-chief 

HILDEBRAND,  JOHN,  Columbia  University 
REESE,  THOMAS,  National  Institutes  of  Health 

Other  faculty,  staff,  and  lecturers 

ALKON,  DANIEL,  National  Institutes  of  Health/Marine  Biological  Laboratory 
ALLEN,  ROBERT,  Dartmouth  College 


68  MARINE  BIOLOGICAL  LABORATORY 

BARLOW,  ROBERT,  Syracuse  University 

BATTELLE,  BARBARA,  National  Institutes  of  Health 

BRANTON,  DANIEL,  Harvard  University 

BURD,  GAIL,  Massachusetts  General  Hospital 

COULTER,  JOSEPH,  University  of  Texas 

CRISTAKIS,  NICHOLAS,  Yale  University 

DOWLING,  JOHN,  Harvard  University 

DUNLAP,  KATHLEEN,  Tufts  Medical  School 

FISCHBACH,  GERALD,  Harvard  Medical  School 

FURSHPAN,  EDWIN,  Harvard  Medical  School 

GELPERIN,  ALAN,  Princeton  University 

GOY,  MICHAEL,  Harvard  Medical  School 

GRAHAM,  WILLIAM,  National  Institutes  of  Health 

HALBEISEN,  JOHANNA 

HALL,  LINDA,  Albert  Einstein  College  of  Medicine 

HUTTNER,  SUSANNE,  University  of  California  at  Los  Angeles 

KANDEL,  ERIC,  Columbia  University  College  of  Physicians  and  Surgeons 

KENT,  KARLA,  Columbia  University 

KRAVITZ,  EDWARD,  Harvard  Medical  School 

LAFRATTA,  JAMES,  Harvard  Medical  School 

LANDIS,  DENNIS,  Massachusetts  General  Hospital 

LANDIS,  STORY,  Harvard  Medical  School 

LASER,  RAY,  Case  Western  Reserve  University 

LICHTMAN,  JEFF,  Harvard  Medical  School 

MANSOUR,  RANDA,  University  of  North  Carolina 

MATSUMOTO,  STEVEN,  Harvard  Medical  School 

MEINERTZHAGEN,  IAN,  Dalhousie  University,  Canada 

MENZEL,  RANDOLF,  Free  University  of  Berlin,  Germany 

NICHOLLS,  JOHN,  Stanford  University 

NISHI,  RAE,  Harvard  Medical  School 

O'CoNNELL,  MAUREEN,  National  Institutes  of  Health 

O'LAGUE,  PAUL,  University  of  California  at  Los  Angeles 

POTTER,  DAVID,  Harvard  Medical  School 

RAHAMIMOFF,  RAMI,  Hebrew  University  Medical  School,  Israel 

RAVIOLA,  ELIO,  Harvard  Medical  School 

REESE,  BONNIE,  National  Institutes  of  Health 

ROBINSON,  DAVID,  Johns  Hopkins  University 

SCHWAB,  MARTIN,  Max  Planck  Institute  for  Psychiatry,  Germany 

SCHWARTZ,  JAMES,  Columbia  University  College  of  Physicians  and  Surgeons 

SHOTTON,  DAVID,  University  of  Oxford,  United  Kingdom 

SMITH,  STEVE,  Yale  University 

VENKATESH,  T.  R.,  Albert  Einstein  College  of  Medicine 

WALROND,  JOHN,  National  Institutes  of  Health 

WIESEL,  TORSTEN,  Harvard  Medical  School 

WILLARD,  MARK,  Washington  University 

WYMAN,  ROBERT,  Yale  University 

ZIGMOND,  RICHARD,  Harvard  Medical  School 

ZUKIN,  SUZANNE,  Albert  Einstein  College  of  Medicine 

Students1 

*BOYARSKY,  GREGORY,  Yale  University 

*CORMIER,  SUSAN,  Clark  University 

*FELS,  GREGOR,  Max  Planck  Institute,  Germany 

*HOSKINS,  SALLY,  University  of  Chicago 

*HUBBARD,  KAREN,  Illinois  Institute  of  Technology 

*MATTOX,  DOUGLAS,  University  of  Texas  Health  Science  Center 


EDUCATIONAL  PROGRAMS  69 

*MENCO,  BERNARD,  University  of  Utrecht,  Netherlands 

*SILBERSTEIN,  LAURA,  University  of  California  at  San  Francisco  Medical  School 

*STEVENS,  LESLIE,  Harvard  University 

*STUENKEL,  EDWARD,  University  of  Hawaii 

*VOLMAN,  SUSAN,  Cornell  University 

*WIELAND,  STEVEN,  Princeton  University 

PHYSIOLOGY 

Instructor -in-chief 

ROSENBAUM,  JOEL,  Yale  University 

Other  faculty,  staff,  and  lecturers 

ACKERS,  GARY,  Johns  Hopkins  University 

ALBRECHT,  GUENTER,  Cold  Spring  Harbor  Labs 

ALEXANDRAKI,  DESPINA,  Harvard  Medical  School 

ALLEN,  ROBERT,  Dartmouth  College 

ALLEWELL,  NORMA,  Wesleyan  University 

ALTMAN,  SID,  Yale  University 

AMOS,  LINDA,  Medical  Research  Council,  United  Kingdom 

BALLINGER,  DENNIS,  Massachusetts  Institute  of  Technology 

BARNARD,  STEVE,  Connecticut  College 

BENNETT,  VANN,  Johns  Hopkins  University 

CHISHOLM,  REX,  Massachusetts  Institute  of  Technology 

CHUA,  NAM  Hi,  Rockefeller  University 

CLEVELAND,  DON,  Johns  Hopkins  University 

COHEN,  CARL,  St.  Elizabeth's  Hospital 

CONDEELIS,  JOHN,  Albert  Einstein  College  of  Medicine 

GRAIN,  WILLIAM,  The  Worcester  Foundation  for  Experimental  Biology 

DENTLER,  WILLIAM,  University  of  Kansas 

DETERRA,  NOEL,  Marine  Biological  Laboratory 

FELSARFELD,  DAN 

GOLDMAN,  ANN,  Northwestern  University  Medical  School 

GOLDMAN,  ROBERT,  Carnegie-Mellon  University 

GOROVSKY,  MARTIN,  University  of  Rochester 

GREENE,  JOHN,  Massachusetts  Institute  of  Technology 

GUARANTE,  LENNY,  Harvard  University 

HEREFORD,  LYNNA,  Brandeis  University 

HOBBIE,  LAWRENCE,  Woods  Hole,  MA 

HUNT,  TIMOTHY,  Cambridge  University,  United  Kingdom 

HUXLEY,  HUGH,  Medical  Research  Council,  United  Kingdom 

INOUE,  SHINYA,  Marine  Biological  Laboratory 

JONES,  JONATHAN,  Northwestern  University  Medical  School 

KABACK,  DAVID,  New  Jersey  Medical  School 

KALFAYAN,  LAURA,  Brandeis  University 

KEDES,  LARRY,  Stanford  University 

KHAN,  HAMID,  Northwestern  University  Medical  School 

KILMARTIN,  JOHN,  Medical  Research  Council,  United  Kingdom 

KAMIYA,  NOBURO,  National  Institute  of  Basic  Biology,  Japan 

LASEK,  RAY,  Case  Western  Reserve  University 

LODISH,  HAVEY,  Massachusetts  Institute  of  Technology 

MAY,  GRER,  Yale  University 

MAYRAND,  SANDY,  Worcester  Foundation  for  Experimental  Biology 

MCCARTHY,  MICHAEL,  Wesleyan  University 

MCKINTOSH,  RICHARD,  University  of  Colorado 


70  1ARINE  BIOLOGICAL  LABORATORY 

MISCHKE,  DIETMAR,       tssachusetts  Institute  of  Technology 

MITCHELL,  DAVID,  University 

MOOSEKER,  MARK,  University 

MORRIS,  RON,  Rutge     University 

MURRY,  ANDRH         Earvard  University 

OSLEY,  MAR1*  Brandeis  University 

_ou,  Massachusetts  Institute  of  Technology 

PEDERSON,  THORU,  Worcester  Foundation  for  Experimental  Biology 
PELHAM,  HUGH,  Carnegie  Institute 

PENMAN,  SHELDON,  Massachusetts  Institute  of  Technology 
PLATT,  TERRY,  Yale  University 
POLLARD,  THOMAS,  Johns  Hopkins  University 
REID,  MARTHA,  Earlham  College 
RUDERMAN,  JOAN,  Harvard  Medical  School 
SILFLOE,  CAROLYN,  Yale  University 
SLOBODA,  ROGER,  Dartmouth  College 
STEINBURG,  ELEANOR,  Oregon  State  University 
STEINBURG,  JULIE,  Macalester  College 
STEINERT,  PETER,  National  Institutes  of  Health 
STEITZ,  JOAN,  Yale  University 
STEITZ,  THOMAS,  Yale  University 
STEPHENS,  RAYMOND,  Marine  Biological  Laboratory 
TALIAN,  JOHN,  Northwestern  University  Medical  School 
TIMASHEFF,  SERGE,  Brandeis  University 
VAHEY,  MARY,  Albert  Einstein  College  of  Medicine 
VAN  HOLDE,  KEN,  University  of  Oregon 
WADSWORTH,  PATRICIA,  Dartmouth  College 
WEINTRAUB,  HAROLD,  Cancer  Research  Institute 
WENSINK,  PIETER,  Brandeis  University 
WINKLER,  MATTHEW,  University  of  California  at  Davis 
ZACHROFF,  ROBERT,  Northwestern  University  Medical  School 

Students1 

*AMOS,  WILLIAM,  University  of  Cambridge,  United  Kingdom 
*BANDZIULIS,  RAYMOND,  Yale  University 
*  BLANK,  PAUL,  Johns  Hopkins  University 
BUHLE,  EMMETT,  Johns  Hopkins  School  of  Medicine 
*CHAMBERS,  CAROLYN,  Xavier  University 
*CHOU,  YING-HAO,  University  of  Virginia 
*DESIMONE,  DOUGLAS,  Dartmouth  College 
*DONIACH,  TABITHA,  University  of  California  at  Santa  Cruz 
*DUNN-COLEMAN,  ELAINE,  University  of  Virginia 
*GREEN,  KATHLEEN,  Washington  University 
*GRIMWADE,  BRIAN,  Yale  University 
*HAGER,  KARL,  Yale  University 
*HERING,  GORDON,  University  of  Wisconsin 
HILDEBRAND,  JOHN,  Stanford  University 
*HOLLINGSWORTH,  NANCY,  Oregon  State  University 
"HOCK,  RICK,  Albert  Einstein  College  of  Medicine 
*JOHNSON,  NANCY,  Harvard  University 
is,  LARRY,  University  of  Virginia 

DSHITERU,  University  of  Tokyo,  Japan 
:CIA,  University  of  Pennsylvania 
Diversity  of  Pennsylvania 

of  California  at  Berkeley 
ij.de  Children's  Research  Hospital 


EDUCATIONAL  PROGRAMS  7 1 

*MROCZKOWSKI,  BARBARA,  University  of  Connecticut 
*RASMUSSEN,  BETH,  University  of  North  Carolina 
*ROBEY,  ELLEN,  University  of  Virginia 
*SNABES,  MICHAEL,  Baylor  College  of  Medicine 
STULTS,  LARRY,  Johns  Hopkins  University 
*UFKES,  SUSAN,  University  of  Massachusetts  at  Amherst 
*WARD,  GARY,  University  of  California  at  San  Diego 

JANUARY 

BASIC  ECOLOGY  AND  MANAGEMENT  OF  RESOURCES 

Instructor s-in-chief 

HOWARTH,  ROBERT,  Marine  Biological  Laboratory 
WOODWELL,  GEORGE,  Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

BREWER,  PETER,  Woods  Hole  Oceanographic  Institution 
CAPUZZO,  JUDITH,  Woods  Hole  Oceanographic  Institution 
CHRISTENSEN,  NORMAN,  Duke  University 
COHEN,  EDWARD,  National  Marine  Fisheries  Service 
COMPTON,  SARAH,  U.  S.  Environmental  Protection  Agency 
FOY,  DOUGLAS,  Conservation  Law  Foundation 
GOLDBERG,  EDWARD,  Scripps  Institution  of  Oceanography 
GRASSLE,  FREDERICK,  Woods  Hole  Oceanographic  Institution 
GROSSLEIN,  MARVIN,  National  Marine  Fisheries  Service 
HEINSELMANN,  MYRON,  U.  S.  Forest  Service 
HINE,  CAPT.  LYNN,  U.  S.  Coast  Guard 
HOBBIE,  JOHN  E.,  Marine  Biological  Laboratory 
HOUGHTON,  RICHARD  A.,  Marine  Biological  Laboratory 
MELILLO,  JERRY  M.,  Marine  Biological  Laboratory 
MURPHY,  EVELYN,  Harvard  University 
PETERSON,  BRUCE  J.,  Marine  Biological  Laboratory 
PISANO,  VICTOR,  TV  Station  38,  Boston 
SANDERS,  HOWARD,  Woods  Hole  Oceanographic  Institution 
SHAVER,  GAIUS  R.,  Marine  Biological  Laboratory 
SNEDECHER,  JAMES,  Anderson  and  Nichols 
SPETH,  Gus,  Council  on  Environmental  Quality 
TEAL,  JOHN,  Woods  Hole  Oceanographic  Institution 
VACCARO,  RALPH,  Woods  Hole  Oceanographic  Institution 

Students 

BROWNAWELL,  BRUCE,  Woods  Hole  Oceanographic  Institution/Massachusetts  Institute  of 

Technology 

DEMUTH,  ROBERT,  University  of  Alabama  in  Birmingham 
ELBE,  UTE,  College  of  Santa  Fe 
GUSMAN,  LINDA,  Creve  Coeur,  MO 
GUTJAHR,  RUTH,  National  Marine  Fisheries  Service 
HAAKONSEN,  HARRY,  Southern  Connecticut  State  College 
KOZAK,  PATRICIA,  University  of  New  Hampshire 
LADERMAN,  RACHEL,  Woods  Hole,  MA 
LIPMAN,  DEBORAH,  Natural  Resources  Defense  Council 
LOWELL,  VICTORIA,  Tufts  University 
LOUGH,  GAYLE,  East  Falmouth,  MA 
MOTT,  RICHARD,  Tulane  University 


72  MARINE  BIOLOGICAL  LABORATORY 

REESE,  DWIGHT,  5          jniversity  of  New  York  at  Stony  Brook 
WEEMS,  JANICE,  Stei     i  University 

BEHAVIOR 

Instructor-in-c 

ATEMA,  JLLLF    3oston  University  Marine  Program/Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

BARLOW,  ROBERT,  Syracuse  University 

BLOUGH,  DONALD,  Brown  University 

BOLLING,  CLAUDE,  Conservation  de  Paris,  France 

BRIDGES,  ROBERT,  Harvard  Medical  School 

BRISBIN,  I.  LEHR,  Savannah  River  Ecology  Program 

BRYANT,  BRUCE,  Boston  University  Marine  Program/Marine  Biological  Laboratory 

CALLARD,  GLORIA,  Harvard  Medical  School 

CAREY,  FRANCIS,  Woods  Hole  Oceanographic  Institution 

CLARK,  CHRISTOPHER,  Rockefeller  University 

DERBY,  CHARLES,  Boston  University  Marine  Program/Marine  Biological  Laboratory 

DETHIER,  VINCENT,  University  of  Massachusetts  at  Amherst 

HDDS,  PEGGY,  University  of  Maryland 

ELGIN,  RANDALL,  Boston  University  Marine  Program/Marine  Biological  Laboratory 

FRANCIS,  ELIZABETH,  Bates  College 

FRAZIER,  JEAN,  Brandeis  University 

GERHARDT,  CARL,  University  of  Missouri 

HAIN,  JAMES,  University  of  Rhode  Island 

HAUSFATER,  GLEN,  Cornell  University 

IRELAND,  LEONARD,  Bermuda  Biological  Station,  Bermuda 

KALMIJN,  ADRIANUS,  Woods  Hole  Oceanographic  Institution 

KAMIL,  AL,  University  of  Massachusetts  at  Amherst 

KREITHEN,  MEL,  University  of  Pittsburgh 

LANGBAUER,  WILLIAM,  C.  V.  Whitney  Laboratory 

LEVINE,  JOSEPH,  Harvard  University 

MOLLER,  PETER,  American  Museum  of  Natural  History 

NYBY,  JOHN,  Lehigh  University 

RISTAU,  CAROLYN,  Rockefeller  University 

STUART,  ALASTAIR,  University  of  Massachusetts  at  Amherst 

SULZMAN,  FRANK,  State  University  of  New  York  at  Binghamton 

SWAIN,  TONY,  Boston  University 

WILCOX,  STIMSON,  State  University  of  New  York  at  Binghamton 

WILLIAMS,  TIMOTHY,  Swarthmore  College 

WILLIAMS,  JANET,  Swarthmore  College 

Students 

BAKER,  TAHIRIH,  Jackson  State  University 
BROWN,  JANICE,  Tougaloo  College 
BRYANT,  DONALD,  Boston  University 
CARACO,  NINA,  Boston  University 
ELLIOTT,  WANDA,  Jackson  State  University 
FERME,  PAOLA,  Boston  University 
FUJITA,  RODNEY,  Boston  University 
GOLDFARB,  SHARI,  Cornell  University 
Moss,  ANTHONY,  Boston  University 
SCHUTRUMPF,  ANDREW,  Northeastern  University 
SEGAL,  YVETTE,  Cornell  University 


EDUCATIONAL  PROGRAMS  73 

COMPARATIVE  PATHOLOGY  OF  MARINE  INVERTEBRATES 
Instructor-in-chief 

BANG,  FREDERIK,  Johns  Hopkins  University 
Other  faculty,  staff,  and  lecturers 

BANG,  BETSY,  Johns  Hopkins  University 

CHANG,  PEI  WEN,  University  of  Rhode  Island 

COOPER,  KEITH,  Thomas  Jefferson  University  Medical  School 

DUCKLOW,  HUGH,  Columbia  University 

HDDS,  KENNETH,  State  University  of  New  York  at  Buffalo 

ELSTON,  RALPH,  Cornell  University 

FARLEY,  AUSTIN,  National  Marine  Fisheries  Service 

LEVIN,  JACK,  Johns  Hopkins  University 

MICHELSON,  EDWARD,  Harvard  University 

PEARCE,  JACK,  National  Marine  Fisheries  Service 

PRENDERGAST,  ROBERT,  Johns  Hopkins  University 

REINISCH,  CAROL,  Harvard  Medical  School 

STRANDBERG,  JOHN,  Johns  Hopkins  University 

SCARBOROUGH,  ANN,  Johns  Hopkins  University 

Students 

ANDERSON,  KENNETH,  University  of  Chicago 

FONTAINE,  ANNE,  Mount  Holyoke  College 

GILLES,  KAY,  University  of  California  at  Santa  Cruz 

KLINGENSMITH,  J.  SCOTT,  University  of  Mississippi  Medical  School 

KOELLE,  REINER,  Eisenhower  College 

LANDY,  RONALD,  University  of  Pennsylvania 

LEONARD,  LESLIE,  Johns  Hopkins  University 

MANIGLIA,  MARY  ANN,  Roosevelt  University 

MCCORMICK-RAY,  M.  GERALDINE,  University  of  Virginia 

ROCK,  ALAN,  University  of  Rhode  Island 

SHIFTLET,  GEORGE,  Erskine  College 

SILVERMAN,  BARRY,  Johns  Hopkins  University 

SPEARS,  CLIFTON,  Dillard  University 

DEVELOPMENTAL  BIOLOGY 
Instructor-in-chief 

EDDS,  KENNETH,  State  University  of  New  York  at  Buffalo 
Other  faculty,  staff,  and  lecturers 

ALBERTINI,  DAVID,  Harvard  Medical  School 

BEGG,  DAVID,  Harvard  Medical  School 

BELL,  EUGENE,  Massachusetts  Institute  of  Technology 

COLEMAN,  ANNETTE,  Brown  University 

COLEMAN,  JOHN,  Brown  University 

FUJIWARA,  KUIGI,  Harvard  Medical  School 

GERBI,  SUSAN,  Brown  University 

GOLD,  BERT,  Tufts  University 

GROSS,  PAUL,  Marine  Biological  Laboratory 

HARRIGAN,  JUNE,  Marine  Biological  Laboratory 

HEIPLE,  JEANNE,  Harvard  University 

HEPLER,  PETER,  University  of  Massachusetts  at  Amherst 


74  MARINE  BIOLOGICAL  LABORATORY 

INOUE,  SHINYA,  ;  Biological  Laboratory 

LINCK,  RICH  .rvard  Medical  School 

Luxz,  DOUGL.A          irine  Biological  Laboratory 

MARCUS,  NANC       ?voods  Hole  Oceanographic  Institution 

MARIN,  FRE  vn  University 

MASER,  Mo  Marine  Biological  Laboratory 

ROSENTHA     ERIC,  Harvard  University 

RUDERMAN,      'AN,  Harvard  Medical  School 

SCHUEL,  HERBERT,  State  University  of  New  York  at  Buffalo 

TAMM,  SIDNEY,  Boston  University/Marine  Biological  Laboratory 

TAYLOR,  D.  LANSING,  Harvard  University 

TROTT,  THOMAS,  Boston  University 

VOGEL,  A.  WAYNE,  Harvard  Medical  School 

YAMIN,  MICHAEL,  Marine  Biological  Laboratory 

Students 

ARCHIBALD,  ASTON,  Clark  College 
BERG,  JOSEPH,  California  State  College 
COLEMAN,  EDWARD,  Texas  Southern  University 
HAY,  BRUCE,  Claremont  Men's  College 
JENKINS,  GAYE,  Dillard  University 
JOHNSON,  DENISE,  Texas  Southern  University 
MENZEL,  CHARLOTTE,  Oberlin  College 
MOULDING,  CHRISTOPHER,  Harvard  University 
NEGUS,  JAMES,  Wofford  College 
PETTIS,  RENEE,  Texas  Southern  University 
SANDERS,  PAMELA,  Texas  Southern  University 
WALKER,  ROSIE,  Tougaloo  College 

NEUROBIOLOGY 

Instructor -in- chief 

ALKON,  DANIEL,  National  Institutes  of  Health/Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

ADELMAN,  WILLIAM,  National  Institutes  of  Health/Marine  Biological  Laboratory 

ATWOOD,  HAROLD,  University  of  Toronto 

BARLOW,  ROBERT,  Syracuse  University 

BRIGHTMAN,  MILTON,  National  Institutes  of  Health 

CONNOR,  JOHN,  University  of  Illinois 

DEFELICE,  Louis,  Emory  University  School  of  Medicine 

DOWLING,  JOHN,  Harvard  University 

FARLEY,  JOSEPH,  Princeton  University 

FEIN,  ALAN,  Boston  University  Medical  School/Marine  Biological  Laboratory 

GOODMAN,  STEVEN,  Marine  Biological  Laboratory 

GOVIND,  C.  K.,  University  of  Toronto,  Canada 

HAROSI,  FERENCE,  Boston  University/Marine  Biological  Laboratory 

JACKLET,  JON,  State  University  of  New  York  at  Albany 

KAPLAN,  EHUD,  Rockefeller  University 

KRAVITZ,  EDWARD,  Harvard  Medical  School 

IZIRIAN,  ALAN,  National  Institutes  of  Health/Marine  Biological  Laboratory 

iHiiNDLER,  I.  IZJA,  National  Institutes  of  Health/Marine  Biological  Laboratory 
1ON,  Harvard  Medical  School 
N,  University  of  Massachusetts 

National  Institutes  of  Health/Marine  Biological  Laboratory 


EDUCATIONAL  PROGRAMS  75 

PAPPAS,  GEORGE,  University  of  Illinois 

PINTO,  LAWRENCE,  Purdue  University 

POTTER,  DAVID,  Harvard  Medical  School 

PRICE,  CHRISTOPHER,  Boston  University 

RASMUSSEN,  HOWARD,  Yale  University  School  of  Medicine 

RAYMOND,  STEPHEN,  Massachusetts  Institute  of  Technology 

RICHARDS,  WILLIAM,  Marine  Biological  Laboratory 

SCHWARTZ,  JAMES,  Columbia  University 

SHEPHERD,  GORDON,  Yale  University  School  of  Medicine 

SHOUKIMAS,  JONATHAN,  National  Institutes  of  Health/Marine  Biological  Laboratory 

SENFT,  STEPHEN,  University  of  Oregon 

STEPHENS,  RAYMOND,  Boston  University  School  of  Medicine/Marine  Biological  Laboratory 

SZUTS,  ETE,  Marine  Biological  Laboratory 

TAMM,  SIDNEY,  Boston  University/Marine  Biological  Laboratory 

THOMPSON,  CHARLES,  University  of  Toronto,  Canada 

WALOGA,  GERALDINE,  Boston  University  School  of  Medicine 

WEISS,  THOMAS,  Massachusetts  Institute  of  Technology 

Students 

BOLDEN,  MARSHA,  Texas  Southern  University 

BROOKS,  CHARLES,  Washington  University 

BRY,  JOHN,  Massachusetts  General  Hospital 

CHING-JU,  CHEN,  University  of  Rhode  Island 

EISELE,  LESLIE,  University  of  Colorado  at  Boulder 

FAMIGLIO,  GREGORY,  Massachusetts  Institute  of  Technology 

FREED,  LAUREL,  University  of  Bridgeport 

GART,  SERGE,  Marlboro  College 

KRIKORIAN,  JACQUELINE,  University  of  Maryland  School  of  Medicine 

LANDRY,  ANNE,  Mount  Holyoke  College 

LAYTON,  BARRY,  The  Montreal  General  Hospital,  Canada 

LYDIC,  RALPH,  Harvard  Medical  School 

NAFTOLOWITZ,  DAVID,  Amherst  College 

NORMAN,  PHILLIPPA,  Tougaloo  College 

PEARLSON,  YALE,  Tufts  University 

ROCHEL,  SARAH,  Roche  Institute  of  Molecular  Biology 

SMITH,  MARVA,  Dillard  University 

ZEBLEY,  ELMER,  New  College 

SHORT  COURSES 

ANALYTICAL  AND  QUANTITATIVE  LIGHT  MICROSCOPY  IN  BIOLOGY,  MEDICINE, 

AND  MATERIALS  SCIENCES 

Instructor-in-chief 

INOUE,  SHINYA,  Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

BELCHER,  ARTHUR,  Venus  Scientific 
CHIASSON,  RICHARD,  Olympus  Corporation  of  America 
COOMBS,  GILLIAN,  Marine  Biological  Laboratory 
ELLIS,  GORDON,  University  of  Pennsylvania 
ENDERS,  REINHARD,  E.  Leitz,  Inc. 
HANAWAY,  WINDHAM,  Colorado  Video 
HAYES,  THOMAS,  University  of  North  Carolina 
HEIPLE,  JEANNE,  Harvard  University 


76  MARINE  BIOLOGICAL  LABORATORY 

HINSCH,  JAN,  E.  Leitz,  Inc. 

IIDA,  HITOSHI,  Hamamatsu  Systems,  Inc. 

KELLER,  ERNST,  Carl  Zeiss,  Inc. 

KLEIFGEN,  JERRY,  DAGE-MTI 

LANGENBACH,  UWE,  Seller  Instruments  (aus  Jena) 

LUTZ,  DOUGLAS,  Marine  Biological  Laboratory 

OLWELL,  PATRICIA,  Seiler  Instruments  (aus  Jena) 

RIKUKAWA,  KATSUJI,  Nikon,  Inc. 

SALMON,  EDWARD,  University  of  North  Carolina 

SAWYER,  WILLIAM,  Carl  Zeiss,  Inc. 

SCHEIER,  KURT,  Nikon,  Inc. 

SEIDLE,  WALTER,  Olympus  Corporation  of  America 

SUSSMAN,  GARY,  Crimson  Camera  Technical  Sales,  Inc. 

TAYLOR,  D.  LANSING,  Harvard  University 

TAYLOR,  RICHARD,  Colorado  Video 

THOMAS,  PAUL,  DAGE-MTI 

WALLACE,  PETER,  Crimson  Camera  Technical  Sales,  Inc. 

Students 

ALDRICH,  RICHARD,  Yale  University  Medical  School 

BAJER,  ANDREW,  University  of  Oregon 

BURGOS,  MARIO,  Harvard  Medical  School 

CHAILLET,  JOHN,  Yale  University  Medical  School 

CHANDLER,  WILLIAM,  Yale  University  Medical  School 

COATES,  THOMAS,  Riley  Hospital  for  Children 

IRVING,  MALCOLM,  Yale  University  Medical  School 

KEITH,  CHARLES,  New  York  University  Medical  Center 

McMEEKiN,  LINDA,  American  Cyanamid  Company 

MILLER,  THOMAS,  Harvard  Medical  School 

ROMERO,  FAUSTINO,  Universidad  Nacional  Autonoma  de  Mexico,  Mexico 

SCHATTEN,  GERALD,  Florida  State  University 

SOKOLOSKI,  JOSEPH,  University  of  Pennsylvania 

WEISS,  GILBERT,  Life  Savers,  Inc. 

BIOLOGICAL  ELECTRON  MICROSCOPY  FOR  TECHNICIANS 

Instructor -in-chief 

MASER,  MORTON,  Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

ANTOL,  JOE,  Carl  Zeiss,  Inc. 

COPELAND,  D.  EUGENE,  Marine  Biological  Laboratory 

GEISER,  ALBERT,  Hahnemann  Medical  Center 

HOUGHTON,  SUSAN,  Woods  Hole,  MA 

SAWYER,  WILLIAM,  Carl  Zeiss,  Inc. 

UYDESS,  IAN,  Carl  Zeiss,  Inc. 

Students 

ARISSIAN,  KOSTADINKA,  Rockefeller  University 
BEETON,  PHYLLIS,  Temple  University  School  of  Medicine 
BOCK,  KITTY,  Columbia  University 
BROWN,  RUTH  ANN,  Washington  State  University 
CAVANAUGH,  COLLEEN,  Marine  Biological  Laboratory 
FERGUSON,  PAULA,  Maine  Medical  Center 


EDUCATIONAL  PROGRAMS  77 

GREEN,  KAREN,  Dillard  University 
KIMBERLY,  PRISCILLA,  University  of  Vermont 
KOEHLER,  INGRID,  Brown  University 
LEMAY,  PETER,  College  of  the  Holy  Cross 
SCHOUN,  VERA,  St.  Vincent  Charity  Hospital 
SIMS,  DAVID,  St.  Vincent  Charity  Hospital 

ELECTRON  MICROSCOPY  IN  THE  BIOLOGICAL  SCIENCES 

Instructors-in-chief 

BOWERS,  BLAIR,  National  Institutes  of  Health 
MASER,  MORTON,  Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

COOMBS,  GILLIAN,  Marine  Biological  Laboratory 
COPELAND,  D.  EUGENE,  Marine  Biological  Laboratory 
HOHMAN,  THOMAS,  National  Institutes  of  Health 
HOUGHTON,  SUSAN,  Woods  Hole,  MA 
PEACHEY,  LEE,  University  of  Pennsylvania 
PORTER,  KEITH,  University  of  Colorado 
WATERBURY,  JOHN,  Woods  Hole  Oceanographic  Institution 
WILLINGHAM,  MARK,  National  Institutes  of  Health 

Students 

ANGELO,  JEAN,  Bowman  Gray  School  of  Medicine 

BATTISTA,  ARTHUR,  New  York  University  Medical  School 

BRYER,  PAMELA,  Bowdoin  College 

EELLS,  THOMAS,  Mary  Imogene  Bassett  Hospital 

FITE,  KATHERINE,  University  of  Massachusetts  at  Amherst 

GALLANT,  PAUL,  National  Institutes  of  Mental  Health 

HALL,  MICHAEL,  University  of  California  School  of  Medicine  at  Los  Angeles 

HESSLER,  ANITA,  University  of  California  at  San  Diego 

JORDAN,  THOMAS,  Dillard  University 

MLADENOV,  PHILIP,  Mount  Allison  University,  Canada 

MURPHY-ULLRICH,  JOANNE,  University  of  Wisconsin 

THOMAS,  JUDITH,  North  Carolina  State  University 

FREEZE-ETCHING  IN  ELECTRON  MICROSCOPY 

Instructor-in-chief 

STEERE,  RUSSELL,  U.  S.  Department  of  Agriculture 

Other  faculty,  staff,  and  lecturers 

COOMBS,  GILLIAN,  Marine  Biological  Laboratory 
DEINAN,  DERMOT,  Polaron  Instruments,  Inc. 
ERBE,  ERIC,  U.  S.  Department  of  Agriculture 
GRAHAM,  WILLIAM,  National  Institutes  of  Health 
RASH,  JOHN,  Colorado  State  University 

Students 

BELL,  BARBARA,  Atlanta  University 

COCKERHAM,  LORRIS,  Armed  Forces  Radiobiology  Research  Institution 

DUDEK,  RONALD,  East  Carolina  University  Medical  School 

GEISER,  ALBERT,  Hahnemann  Medical  College  and  Hospital 


78  MARINE  BIOLOGICAL  LABORATORY 

KANNAN,  MATHUR,  McMaster  Medical  Centre,  Canada 
NUTTALL,  ROBERT,  Emory  University 
ROSLANSKY,  PRISCILLA,  Marine  Biological  Laboratory 
RYERSE,  JAN,  St.  Louis  University  School  of  Medicine 
WATSON,  GLEN,  Florida  State  University 

MARICULTURE:  CULTURE  OF  MARINE  INVERTEBRATES 
FOR  RESEARCH  PURPOSES 

Instructor-in-chief 

BERG,  CARL,  Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

CAPUZZO,  JUDITH,  Woods  Hole  Oceanographic  Institution 

CAPO,  THOMAS,  Marine  Biological  Laboratory 

DEAN,  DAVID,  University  of  Maine 

DOYLE,  ROGER,  Dalhousie  University,  Canada 

EARLY,  GREGORY,  New  England  Aquarium 

ELSTON,  RALPH,  Cornell  University 

FUJITA,  RODNEY,  Marine  Biological  Laboratory 

GARIBALDI,  Louis,  New  England  Aquarium 

GOLDMAN,  JOEL,  Woods  Hole  Oceanographic  Institution 

GUILLARD,  ROBERT,  Woods  Hole  Oceanographic  Institution 

HARRIGAN,  JUNE,  Marine  Biological  Laboratory 

HUGHES,  JOHN,  Massachusetts  State  Lobster  Hatchery 

HIXON,  RAYMOND,  Marine  Biomedical  Institute 

MANN,  ROGER,  Woods  Hole  Oceanographic  Institution 

MARCUS,  NANCY,  Woods  Hole  Oceanographic  Institution 

Students 

AGRAZ,  FERNANDO,  State  Fisheries  Laboratory,  Mexico 
ARCHER,  VERNON,  Jackson  State  University 
CASTANARES,  ERIC,  Centre  de  Ciencias  del  Mar,  Mexico 
CARDELLI,  LINDA,  King  James  Shrimp  Company 
CHANG,  ERNEST,  Bodega  Marine  Laboratory 
COLLINS,  MARGERET,  Howard  University 
DIOGUARDI,  PAUL,  Hackettstown,  NJ 
DURFEE,  WAYNE,  University  of  Rhode  Island 
GIBSON,  BARBARA,  University  of  Rhode  Island 
IGELSRUD,  DON,  The  University  of  Calgary,  Canada 
MONTOYA,  HAYDEE,  University  of  Kansas 
OLSON-MOORE,  EDWARD,  Sea  World's  Shark  Institute 
STICKLE,  WILLIAM,  Louisiana  State  University 
ZIMMERMAN,  JOHN,  Yale  School  of  Medicine 

OPTICAL  MICROSCOPY  AND  IMAGING  IN  THE  BIOMEDICAL  SCIENCES 

Instructor-in-chief 

ALLEN,  ROBERT,  Dartmouth  College 

Other  faculty,  staff,  and  lecturers 

ALLEN,  NINA,  Dartmouth  College 
BADY,  MICHAEL,  Nikon,  Inc. 
BROWN,  DOUGLAS,  Dartmouth  College 


EDUCATIONAL  PROGRAMS  79 

DECKER,  MEL,  Opti-Quip,  Inc. 

GUNDLACH,  HEINZ,  Carl  Zeiss,  Inc.,  Germany 

HANSEN,  ERIC,  Dartmouth  College 

HAYDEN,  JOHN,  Dartmouth  College 

MORGAN,  ERICH,  Marine  Biological  Laboratory 

INOUE,  SHINYA,  Marine  Biological  Laboratory 

KELLER,  ERNST,  Carl  Zeiss,  Inc. 

KENEALY,  JAMES,  Hamamatsu  Systems,  Inc. 

MALDARI,  MARIO,  Hamamatsu  Systems,  Inc. 

PRESLEY,  PHIL,  Carl  Zeiss,  Inc. 

POST,  NICK,  Nikon,  Inc. 

PHILLIPS,  RICK,  Olympus  Corporation  of  America 

SCHEIER,  KURT,  Nikon,  Inc. 

SCOTT,  ERIC,  Venus  Scientific 

SCOTT,  MARTIN,  Eastman  Kodak  Company 

TAYLOR,  MARCIA,  Olympus  Corporation  of  America 

VAUGHAN,  WILLIAM,  Vickers  Instruments 

WEBB,  WATT,  Cornell  University 

WICK,  ROBERT,  Carl  Zeiss,  Inc. 

WONG,  LENORA,  Dartmouth  College 

Students 

ALLRED,  LAWRENCE,  S.  C.  Johnson  &  Son 

BOWEN,  WILLIAM,  University  of  Arkansas  at  Little  Rock 

BRADY,  SCOTT,  Case  Western  Reserve  University 

CHENEY,  DARWIN,  St.  Elizabeth's  Hospital 

COCKERHAM,  LORRIS,  Armed  Forces  Radiobiology  Research  Institute 

FISHER,  RICHARD,  National  Institutes  of  Health 

GILPIN,  R.  R.,  Clarkson  College  of  Technology 

KACHAR,  BECHARA,  National  Institutes  of  Health 

LINDSEY,  JAMES,  University  of  California  at  San  Diego  School  of  Medicine 

PALMER,  ELIZABETH,  Instituto  Politecnico  Nacional,  Mexico 

PAOLINI,  PAUL,  San  Diego  State  University 

PESCH,  GERALD,  U.  S.  Environmental  Protection  Agency 

RINNERTHALER,  GOTTFRIED,  Institute  of  Molecular  Biology,  Austria 

SMITH,  RICHARD,  University  of  Alberta,  Canada 

SIMPSON,  MARGARET,  Sweet  Briar  College 

PROTEIN  ANALYSIS  BY  POLYACRYLAMIDE  GEL  ELECTROPHORESIS 

Instructor-in-chief 

STEPHENS,  RAYMOND,  Marine  Biological  Laboratory 

Other  faculty,  staff,  and  lecturers 

HERLANDS,  Louis,  The  Rockefeller  University 
HORGAN,  ERICH,  Marine  Biological  Laboratory 
LINCK,  RICHARD,  Harvard  Medical  School 
PIPERNO,  GIANNI,  The  Rockefeller  University 
ZWEIDLER,  ALFRED,  The  Institute  for  Cancer  Research 

Students 

CURFMAN,  GREGORY,  Dartmouth-Hitchcock  Medical  Center 
DiSTEFANO,  JOHN,  Northport  Veterans  Administration  Medical  Center 
DOUGHERTY,  EDWARD,  United  States  Department  of  Agriculture 
FERRIS,  V.  R.,  Purdue  University 


80  MARINE  BIOLOGICAL  LABORATORY 

FLOYD,  CARL,  Morehouse  College 

FLOYD,  PATRICIA,  Morehouse  College 

GREBNER,  EUGENE,  Thomas  Jefferson  University 

HARTZELL,  CHARLES,  Alfred  I.  duPont  Institute 

LOTSHAW,  DAVID,  State  University  of  New  York  at  Albany 

MA,  NANCY,  Harvard  Medical  School 

SEVERSON,  ARLEN,  University  of  Minnesota  at  Duluth  School  of  Medicine 

SHIPPER,  KATHLEEN,  Milton  S.  Hershey  Medical  Center 

TASSAVA,  ROY,  Ohio  State  University 

TUCKER,  ROBERT,  Johns  Hopkins  Oncology  Center 

VERRETT,  JOYCE,  Dillard  University 

ZIMMER,  DANNA,  Baylor  College  of  Medicine 

QUANTITATIVE  ANALYSIS  OF  ELECTRON  MICROGRAPHS 

Instructor --in-chief 

PEACHEY,  LEE,  University  of  Pennsylvania 

Other  faculty,  staff,  and  lecturers 

BUSCHMANN,  ROBERT,  Veterans  Administration  at  Chicago 
HASELGROVE,  JOHN,  University  of  Pennsylvania 
HORGAN,  ERICH,  Marine  Biological  Laboratory 
PALMER,  LARRY,  University  of  Pennsylvania 

Students 

BENSHALOM,  GADI,  National  Institutes  of  Health 

BOTTICELLI,  LAWRENCE,  Stanford  Medical  School 

BURNETT,  PAUL,  Polaroid  Corporation 

CONTOS,  NICHOLAS,  Florida  State  University 

FALLON,  JOHN,  Massachusetts  General  Hospital 

FESER,  LEE,  Armed  Forces  Radiobiology  Research  Institute 

GREGG,  MARYBELLE,  NCI — Frederick  Cancer  Research  Facility 

HOFTIEZER,  VIRGIL,  Indiana  University  School  of  Medicine 

JONES,  CARL,  University  of  Illinois  Medical  Center 

KORTE,  GARY,  Montefiore  Hospital  &  Medical  Center 

LEE,  SHEU-LING,  New  England  Medical  Center  Hospital 

LEMPKA,  TIM,  Vanderbilt  University  Medical  School 

NUNZI,  MARIA,  FIDIA  Research  Laboratories,  Italy 

PALATINI,  DENNIS,  American  Cyanamid  Company 

SCHOTLAND,  DONALD,  Hospital  of  the  University  of  Pennsylvania 

WILSON,  KATHRYN,  Indiana  University  and  Purdue  University  of  Indianapolis 

SMALL  COMPUTERS  IN  BIOMEDICAL  RESEARCH 

Instructor-in-chief 

PALMER,  LARRY,  University  of  Pennsylvania 

Other  faculty,  staff,  and  lecturers 

BLAIR,  RICHARD,  Dartmouth  Medical  School 
DEZMELYK,  ROBERT,  Lab  Computer  Systems,  Inc. 
HORGAN,  ERICH,  Marine  Biological  Laboratory 
PEACHEY,  LEE,  University  of  Pennsylvania 


EDUCATIONAL  PROGRAMS  81 


Students 


AIGNER,  THOMAS,  University  of  Chicago 

ARONSON,  FRANK,  Downstate  Medical  Center 

HOFTIEZER,  VIRGIL,  Indiana  University  School  of  Medicine 

KAUER,  JOHN,  Yale  University 

KENNEDY,  BRIAN,  Yale  University 

MURPHY,  E.  HAZEL,  Medical  College  of  Pennsylvania 

MURRAY,  MARION,  Medical  College  of  Pennsylvania 

NUNZI,  MARIA,  FIDIA  Research  Laboratories,  Italy 

OBAID,  ANA  LIA,  University  of  Pennsylvania 

OLIVER,  JOSEPH,  Medical  College  of  Pennsylvania 

RHODES,  ROBERT,  Case  Western  Reserve  University 

SALZBERG,  BRIAN,  University  of  Pennsylvania 

WILCOX,  R.  STIMSON,  State  University  of  New  York  at  Binghamton 

WILKES,  MARY,  Medical  College  of  Pennsylvania 

ZINGARO,  GLORIA,  Medical  College  of  Pennsylvania 


XI.  RESEARCH  AND  TRAINING  PROGRAMS 

SUMMER 

PRINCIPAL  INVESTIGATORS 

ADAMS,  PAUL  R.,  University  of  Texas  Medical  Branch 

ALBERTE,  RANDALL  S.,  University  of  Chicago 

ALLEN,  ROBERT  D,  Dartmouth  College 

ARMSTRONG,  CLAY  M.,  University  of  Pennsylvania 

ARNOLD,  JOHN  M.,  Kewalo  Marine  Laboratory 

BARISH,  MICHAEL  E.,  University  of  California  at  Los  Angeles 

BARLOW,  ROBERT  B.,  Syracuse  University 

BARTLETT,  GRANT,  Lab  for  Comparative  Biochemistry 

BEAUGE,  Luis,  Institution  Investigacion  Medica,  Argentina 

BEGG,  DAVID  A.,  Harvard  Medical  School 

BELL,  WAYNE  H.,  Hamilton  College 

BENNETT,  MICHAEL  V.  L.,  Albert  Einstein  College  of  Medicine 

BEZANILLA,  FRANCISCO,  University  of  California  at  Los  Angeles 

BODZNICK,  DAVID,  Wesleyan  University 

BORGESE,  THOMAS  A.,  CUNY,  Lehman  College 

BOYER,  BARBARA  C,  Union  College 

BRENCHLEY,  GAYLE  A.,  University  of  California  at  Irvine 

BRODWICK,  MALCOLM  S.,  University  of  Texas  Medical  Branch 

BROWN,  JOEL  E.,  SUNY,  Stony  Brook 

BRUNKEN,  WILLIAM  J.,  New  York  University  Medical  Center 

BULLOCK,  JAMES  O.,  Rush  Presbyterian-Saint  Luke's  Medical  Center 

BURDICK,  CAROLYN  J.,  Brooklyn  College 

BURGER,  MAX  M.,  University  of  Basel,  Switzerland 

CAHALAN,  MICHAEL  D.,  University  of  California  at  Irvine 

CARIELLO,  Lucio,  Stazione  Zoologica  di  Napoli,  Italy 

CHAD,  JOHN  E.,  University  of  California  at  Los  Angeles 

CHANG,  DONALD  C.,  Baylor  College  of  Medicine 

CHAPPELL,  RICHARD  L.,  CUNY,  Hunter  College 

CHARLTON,  MILTON  P.,  Ohio  University 

COHEN,  LAWRENCE  B.,  Yale  University  School  of  Medicine 


82  MARINE  BIOLOGICAL  LABORATORY 

COHEN,  WILLIAM  D.,  CUNY,  Hunter  College 

COOPERSTEIN,  SHERWIN  J.,  University  of  Connecticut  Health  Center 

DEKIN,  MICHAEL  S.,  SUNY,  Albany 

DENTLER,  WILLIAM  L.,  University  of  Kansas 

DETERRA,  NOEL,  Hahnemann  Medical  College  and  Hospital 

DEWEER,  PAUL  J.,  Washington  University  School  of  Medicine 

DOWLING,  JOHN  E.,  Harvard  University 

DUBOIS,  ARTHUR,  John  B.  Pierce  Foundation  Lab 

EATON,  DOUGLAS  C.,  University  of  Texas  Medical  Branch 

ECKERT,  ROGER,  University  of  California  at  Los  Angeles 

FARMANFARMAIAN,  A.,  Rutgers  University 

FERNANDEZ,  JUILO,  University  of  California  at  Los  Angeles 

FINGER,  THOMAS  E.,  University  of  Colorado 

FISHMAN,  HARVEY  M.  University  of  Texas  Medical  Branch 

FRENCH,  ROBERT  J.,  University  of  Maryland 

FRESCHI,  JOSEPH  E.,  AFRRI 

FROHLICH,  AMALIE,  Dalhousie  University,  Canada 

FUSSELL,  CATHARINE  P.,  Pennsylvania  State  University 

GILBERT,  DANIEL  L.,  National  Institutes  of  Health 

GLANZMAN,  DAVID  L.,  University  of  California  at  Los  Angeles 

GRAF,  WERNER  M.,  New  York  University  Medical  Center 

GROSCH,  DANIEL  S.,  North  Carolina  State  University 

GUERRIER,  PIERRE,  Station  Biologique,  Roscoff,  France 

HAIMO,  LEAH  T.,  University  of  California  at  Riverside 

HARDING,  CLIFFORD  V.,  Kresge  Eye  Institute 

HASCHEMEYER,  AUDREY  E.  V.,  CUNY,  Hunter  College 

HERNANDEZ-NICAISE,  MARI  Luz,  University  Claude  Bernard,  France 

HEUSER,  JOHN,  Washington  University  School  of  Medicine 

HIGHSTEIN,  STEPHEN  M.,  Albert  Einstein  College  of  Medicine 

HILL,  SUSAN  D.,  Michigan  State  University 

HINCH,  GERTRUDE  W.,  University  of  Southern  Florida 

HOFFMANN,  RICHARD  J.,  Iowa  State  University 

HOGAN,  JAMES  C.,  Yale  University 

HOSKIN,  FRANCIS  C.  G.,  Illinois  Institute  of  Technology 

HUMPHREYS,  TOM,  Kewalo  Marine  Laboratory 

HUNT,  R.  KEVIN,  Johns  Hopkins  University 

ILAN,  JOSEPH,  Case  Western  Reserve  University 

INGOGLIA,  NICHOLAS  A.,  New  Jersey  Medical  School 

IRELAND,  LEONARD,  Bermuda  Biological  Station,  England 

JAMES-KRACK.E,  MARILYN,  Washington  University  School  of  Medicine 

JOSEPHSON,  R.  K.,  University  of  California  at  Irvine 

JOYNER,  RONALD  W.,  University  of  Iowa 

KAMINER,  BENJAMIN,  Boston  University  School  of  Medicine 

KAMIYA,  NOBURO,  National  Institute  for  Basic  Biology,  Okazaki,  Japan 

KAO,  C.  Y.,  SUNY,  Downstate  Medical  Center 

KILDUFF,  THOMAS,  Stanford  University 

KIRSCH,  GLENN  E.,  Rutgers  University 

KOEHL,  MIMI  A.  R.,  University  of  California  at  Berkeley 

KUSANO,  KIYOSHI,  Illinois  Institution  of  Technology 

LANDOWNE,  DAVID,  University  of  Miami 

LASER,  RAYMOND,  Case  Western  Reserve  University 

LAUFER,  HANS,  University  of  Connecticut 

LEE,  JOHN  J.,  CUNY,  City  College 

LEVIN,  JACK,  Johns  Hopkins  Hospital 

LIPICKY,  RAYMOND  J.,  National  Institutes  of  Health 

LLANO,  ISABEL,  University  of  California  at  Los  Angeles 


RESEARCH  AND  TRAINING  PROGRAMS  83 

LLINAS.  R.,  New  York  University  Medical  Center 

LOEWENSTEIN,  WERNER  R.,  University  of  Miami  School  of  Medicine 

LONGO,  FRANK  J.,  University  of  Iowa 

LYTTLE,  C.  RICHARD,  University  of  Pennsylvania 

MAGLOTT,  DONNA  R.,  Howard  University 

MATSUMURA,  FUMIO,  Michigan  State  University 

MAUZERALL,  DAVID,  Rockefeller  University 

McKiNNEY,  LESLIE  C.,  Washington  University 

MEHAFFEY,  LEATHEM,  Vassar  College 

METUZALS,  J.,  University  of  Ottawa,  Canada 

METZ,  CHARLES,  University  of  Miami 

MITCHELL,  RALPH,  Harvard  University 

MOORE,  JOHN  W.,  Duke  University  Medical  Center 

MORRELL,  FRANK,  Rush  Medical  College 

MULLINS,  L.  J.,  University  of  Maryland  School  of  Medicine 

NAGEL,  RONALD  L.,  Albert  Einstein  College  of  Medicine 

NARAHASHI,  TOSHIO,  Northwestern  University  Medical  School 

NELSON,  LEONARD,  Medical  College  of  Ohio 

NOE,  BRYAN  D.,  Emory  University 

NORTHCUTT,  R.  GLENN,  University  of  Michigan 

O'MELIA,  ANNE  F.,  George  Mason  University 

OXFORD,  GERRY  S.,  University  of  North  Carolina 

PAPPAS,  GEORGE  D.,  University  of  Illinois  College  of  Medicine 

PERSON,  PHILIP,  Veterans  Administration  Medical  Center 

PIERCE,  SIDNEY  K.,  University  of  Maryland 

PRZYBYLSKI,  RONALD  J.,  Case  Western  Reserve  University 

REBHUN,  LIONEL  L,  University  of  Virginia 

REYNOLDS,  GEORGE  T.,  Princeton  University 

RICKLES,  FREDERICK  R.,  University  of  Connecticut  Health  Center 

RIPPS,  HARRIS,  New  York  University  School  of  Medicine 

Ross,  WILLIAM  N.,  New  York  Medical  College 

RUDERMAN,  JOAN,  Harvard  Medical  School 

RUSSELL,  JOHN  M.,  University  of  Texas  Medical  Branch 

RUSTAD,  RONALD  C.,  Case  Western  Reserve  University 

SALAMA,  GUY,  University  of  Pittsburgh  School  of  Medicine 

SALMON,  EDWARD  D.,  University  of  North  Carolina 

SALZBERG,  BRIAN  M.,  University  of  Pennsylvania  School  of  Dental  Medicine 

SANGER,  JOSEPH  W.,  University  of  Pennsylvania  School  of  Medicine 

SCHUEL,  HERBERT,  SUNY,  Buffalo 

SCHUETZ,  ALLEN  W.,  Johns  Hopkins  University 

SCHWAB,  WALTER  E.,  Virginia  Polytechnic  Institute  and  State  University 

SCOFIELD,  VIRGINIA  LEE,  Stanford  University  School  of  Medicine 

SEGAL,  SHELDON  J.,  Rockefeller  Foundation 

SMITH,  STEPHEN  J.,  Yale  University  Medical  School 

SPECK,  WILLIAM  T.,  Case  Western  Reserve  University 

SPEIGEL,  EVELYN,  Dartmouth  College 

SPEIGEL,  MELVIN,  Dartmouth  College 

STEPHENS,  PHILIP  J.,  Villanova  University 

STETTEN,  MARJORIE  R.,  National  Institutes  of  Health 

STUART,  ANN  E.,  University  of  North  Carolina 

STUNKARD,  HORACE,  American  Museum  of  Natural  History 

SZAMIER,  R.,  BRUCE,  Massachusetts  Eye  and  Ear  Infirmary 

SZENT-GYORGYI,  ANDREW  G.,  Brandeis  University 

TASAKI,  ICHIJI,  National  Institutes  of  Health 

TAYLOR,  ROBERT  E.,  National  Institutes  of  Health 

TELZER,  BRUCE  R.,  Pomona  College 


84  MARINE  BIOLOGICAL  LABORATORY 

TRINKAUS,  JOHN  P.,  Yale  University 

TROLL,  WALTER,  New  York  University  Medical  Center 

TROXLER,  ROBERT,  Boston  University  School  of  Medicine 

TYTELL,  MICHAEL,  Bowman  Gray  School  of  Medicine 

VANDENBERG,  CAROL  A.,  University  of  California  at  La  Jolla 

VIZA,  DIMITRI,  Faculte  de  Medecine,  France 

WALLACE,  ROBIN,  Oak  Ridge  National  Laboratory 

WARREN,  LEONARD,  Wistar  Institute 

WATSON,  WINSOR  H.,  University  of  New  Hampshire 

WEIDNER,  EARL,  Louisiana  State  University 

WEISSMANN,  GERALD,  New  York  University  Medical  Center 

WHITTAKER,  J.,  RICHARD,  Wistar  Institute 

WORTHINGTON,  C.  R.,  Carnegie-Mellon  University 

ZIGMAN,  SEYMOUR,  University  of  Rochester  School  of  Medicine 

ZUCKER,  ROBERT  S.,  University  of  California 

LIBRARY  READERS 

ADELBERG,  EDWARD,  Yale  University 

ALLEN,  GARLAND  E.,  Washington  University 

ANDERSON,  EVERETT,  Harvard  Medical  School 

ANDRUS,  WILLIAM,  Pomona  College 

APOSHIAN,  H.  V.,  University  of  Arizona 

BAER,  ADELA  S.,  San  Diego  State  University 

BEAN,  CHARLES  P.,  General  Electric  Company 

BECKER,  FREDERICK  F.,  Texas  Medical  Center 

BEIDLER,  L.  M.,  Florida  State  University 

BELL,  EUGENE,  Massachusetts  Institution  of  Technology 

BENSAM,  ARLENE  O.,  M.E.R.I.T.  Fund  Inc. 

BIRNSTIEL,  MAX,  Institution  for  Molecularbiologie,  Switzerland 

BOURNE,  DONALD,  Marine  Biological  Laboratory 

BROWN,  FRANK  A.,  JR.,  Marine  Biological  Laboratory 

BUCK,  JOHN,  National  Institutes  of  Health 

CANDELAS,  GRACIELA  C.,  University  of  Puerto  Rico 

CARLSON,  FRANCIS  D.,  Johns  Hopkins  University 

CARRIERS,  RITA  M.,  New  York  University,  Downstate  Medical  Center 

CAWLING,  V.  F.,  Falmouth,  MA 

CHINARD,  FRANCIS  P.,  New  Jersey  Medical  School 

CLARK,  ARNOLD,  University  of  Delaware 

COBB,  JEWEL  P.,  Rutgers  University,  Douglass 

COHEN,  MAYNARD  M.,  Rush-Presbyterian  St.  Lukes  Medical  Center 

COHEN,  SEYMOUR,  SUNY,  Stony  Brook 

COLE,  JONATHAN,  Cornell  University 

COLLIER,  JACK  R.,  Brooklyn  College 

COLWIN,  ARTHUR  L.,  Key  Biscayne,  FL 

COLWIN,  LAURA  H.,  Key  Biscayne,  FL 

DARDEN,  LINDLEY,  University  of  Maryland 

DETTBARN,  WOLF  D.,  Vanderbilt  University  School  of  Medicine 

DOLINS,  MERELYN,  Rutgers  Medical  School 

EBERT,  JAMES  D.,  Carnegie  Institution  of  Washington 

EDDS,  LOUISE  L.,  Ohio  University 

EDER,  HOWARD  A.,  Albert  Einstein  College  of  Medicine 

ELLISON,  REBECCA  P.,  SUNY,  Purchase 

FADEM,  BARBARA,  New  Jersey  Medical  School 

FEINGOLD,  DAVID  S.,  Tufts  New  England  Medical  School 

FELDMAN,  SUSAN  C.,  New  Jersey  Medical  School 


RESEARCH  AND  TRAINING  PROGRAMS  85 

FISHER,  SAUL  H.,  New  York  University  School  of  Medicine 

FUTRELLE,  ROBERT  P.,  University  of  Illinois 

GABRIEL,  MORDECAI  L.,  Brooklyn  College 

GALATZER-LEVY,  ROBERT  M.,  University  of  Chicago 

GERMAN,  JAMES  L.,  Ill,  New  York  Blood  Center 

GOLDMAN,  ROBERT  D.,  Carnegie-Mellon  University 

GOLDSTEIN,  MOISE  H.,  JR.,  Johns  Hopkins  University 

GRANT,  PHILIP,  University  of  Oregon 

GROSSMAN,  ALBERT,  New  York  University  Medical  Center 

GUTTMAN,  RITA,  SUNY,  Brooklyn  College 

HALL,  ROBERT,  Nantucket  High  School 

HANDLER,  PHILIP,  National  Academy  of  Science 

HAUBRICH,  ROBERT,  Dension  University 

HAUGAARD,  NIELS,  University  of  Pennsylvania 

HELLMAN,  HAL,  Leona,  NJ 

HILL,  RICHARD  W.,  Michigan  State  University 

HILL,  ROBERT  B.,  University  of  Rhode  Island 

HUFNAGEL,  LINDA,  University  of  Rhode  Island 

INOUE,  SHINYA,  Marine  Biological  Laboratory 

ISENBERG,  IRVIN,  Oregon  State  University 

ISSIDORIDES,  MARIETTA  R.,  University  of  Athens,  Greece 

JONAS,  ALBERT  M.,  Tufts  University  School  of  Veterinary  Medicine 

KALTENBACK,  JANE,  Mount  Holyoke  College 

KANE,  ROBERT  E.,  University  of  Hawaii 

KASS-SIMON,  GABRIELE,  University  of  Rhode  Island 

KIRSCHENBAUM,  D.,  SUNY,  Downstate  Medical  Center 

KRAVITZ,  EDWARD,  Harvard  Medical  School 

LADERMAN,  AIMLEE,  Smithsonian  Institution 

LAZAROW,  PAUL,  Rockefeller  University 

LEIGHTON,  JOSEPH,  Medical  College  of  Pennsylvania 

LERMAN,  SIDNEY,  Emory  University 

LESTER,  ROGER,  University  of  Texas  Medical  School 

LEVINE,  RACHMIEL,  City  of  Hope  Medical  Center 

LICHTENSTEIN,  LAWRENCE,  Johns  Hopkins  University  School  of  Medicine 

LOCKWOOD,  ARTHUR,  University  of  North  Carolina 

LORAND,  LASZLO,  Northwestern  University 

MARINE  RESEARCH 

MAUTNER,  HENRY,  Tufts  University  School  of  Medicine 

MAY,  SHELDON  W.,  Georgia  Institute  of  Technology 

McCANN-CoLLiER,  MARJORIES,  St.  Peter's  College 

MINKE,  WILBER,  J.,  Terre  Haute,  IN 

MITCHELL,  JAMES  B.,  Moravian  College 

MIZELL,  MERLE,  Tulane  University 

MONROY,  ALBERTO,  Stazione  Zoologica  di  Napoli,  Italy 

MOOG,  FLORENCE,  Washington  University 

MORSE,  STEPHEN  S.,  Rutgers  University 

NICKELMANN,  SKJOLD  N.,  University  of  California 

O'DONNELL,  JEFF,  Marine  Biological  Laboratory 

OLINS,  DONALD  E.,  University  of  Tennessee 

OLSON,  ROBERT,  St.  Louis  University  School  of  Medicine 

O'RAND,  ANGELA  M.,  Duke  University 

OSCHMAN,  JAMES,  Marine  Biological  Laboratory 

PATON,  DAVID,  Gray  Seal  Research  Project,  Vineyard  Haven,  MA 

PEARLMAN,  ALAN  L.,  Washington  University  School  of  Medicine 

PRUSCH,  ROBERT  D.,  Rhode  Island  College 

QUIGLEY,  JAMES  P.,  SUNY,  Downstate  Medical  Center 


86  MARINE  BIOLOGICAL  LABORATORY 

RANKIN,  MARY  ANN,  University  of  Texas 

REINER,  JOHN  M.,  Albany  Medical  College 

RICE,  ROBERT  V.,  Carnegie  Mellon  University 

RiCH-McCov,  Louis,  Lamont  Geological  Observatory 

ROTH,  JAY  S.,  University  of  Connecticut 

ROWLAND,  LEWIS  P.,  Neurological  Institution 

RUSHFORTH,  NORMAN  B.,  Case  Western  University 

RUSSELL-HUNTER,  W.  D.,  Syracuse  University 

SAGE,  LINDA  C,  University  of  Missouri 

SAGE,  MARTIN,  University  of  Missouri,  St.  Louis 

SAGER,  RUTH,  Sidney  Farber  Cancer  Institute 

SALTZMAN,  MOLLIE,  New  York,  NY 

SAUNDERS,  JOHN  W.,  State  University  of  New  York,  Albany 

SCHWARTZ,  MARTIN,  University  of  Maryland 

SHEMIN,  DAVID,  Northwestern  University 

SHEPARD,  FRANK,  Deep  Sea  Research 

SHEPRO,  DAVID,  Boston  University 

SHERMAN,  IRWIN  W.,  University  of  California,  Riverside 

SIMPSON,  MARGARET,  Sweet  Briar  College 

SISSENWINE,  ILLENE,  Deep  Sea  Research 

SOLOMON,  DENNIS,  Marine  Biological  Laboratory 

SONNENBLICK,  B.  P.,  Rutgers  University 

SPECTOR,  ABRAHAM,  College  of  Physicians  and  Surgeons 

STAFFORD,  WALTER  F.,  Boston  Biomedical  Research  Institution 

STEPHENS,  MICHAEL  J.,  Rutgers  University 

TASHIRO,  JAY  S.,  Kenyon  College 

TRACER,  WILLIAM,  Rockefeller  University 

TWEEDELL,  KENYON  S.,  University  of  Notre  Dame 

VIDAVER-COHEN,  DORIS,  Rush  University 

VIZA,  DIMITRI,  Faculte  de  Medecine,  France 

WAINIO,  WALTER,  Rutgers  University 

WASSERMAN,  R.  H.,  New  York  State  College  of  Veterinary  Medicine 

WATERS,  ROBERT,  Rockefeller  University 

WEBB,  H.  MARGUERITE,  Goucher  College 

WEINBERG,  ERIC,  University  of  Pennsylvania 

WHEELER,  GEORGE  E.,  Brooklyn  College 

WILBER,  CHARLES  G.,  Colorado  State  University 

WILSON,  THOMAS  H.,  Harvard  Medical  School 

WILTSHIRE,  MARK  E.,  Kenyon  College 

WITTENBERG,  BEATRICE,  Albert  Einstein  College  of  Medicine 

WITTENBERG,  JONATHAN,  Albert  Einstein  College  of  Medicine 

YOUNG,  DAVID,  University  of  Melbourne,  Australia 

Yow,  FRANK  W.,  Kenyon  College 

ZACKS,  SUMNER  I.,  Miriam  Hospital 

OTHER  RESEARCH  PERSONNEL 

ADEYEMO,  C.,  Rockefeller  University 

ALDRICH,  RICHARD,  Yale  University  School  of  Medicine 

ALTAMIRANO,  ANIBAL,  Institute  Ferreyra,  Argentina 

AMELAR,  SUSANNA,  New  York  City,  NY 

ANDERSON,  PETER  A.  V.,  Whitney  Marine  Laboratories 

ANSUBEL,  F.  M.,  Harvard  University 

ANTONELLIS,  BLENDA,  Falmouth,  MA 

ARABIAN,  MARY  M.,  Worcester  State  College 

ARANOW,  CYNTHIA,  New  York  University  Medical  Center 


RESEARCH  AND  TRAINING  PROGRAMS  87 

ASANUMA,  NAOKAZU,  Illinois  Institute  of  Technology 

ASHLEY,  MARY,  Kenyon  College 

ASPINALL,  TONI,  Hunter  College 

ATWOOD,  H.  L.,  University  of  Toronto,  Canada 

AUGUSTINE,  GEORGE,  University  of  California,  Los  Angeles 

BAKER,  ROBERT,  New  York  University  Medical  Center 

BARKLEY,  JOHN  J.,  Environmental  Sciences  Center 

BARTELT,  DIANA  C.,  Hunter  College,  CUNY 

BAUER,  G.  ERIC,  University  of  Minnesota 

BELANGER,  ANN  M.,  Emmanuel  College 

BERES,  LINDA  S.,  University  of  California,  Los  Angeles 

BOOKMAN,  RICHARD,  University  of  Pennsylvania 

BORGESE,  JOAN,  Pace  University 

BORON,  W.  F.,  Yale  University 

BOYLE,  MARY  B.,  Yale  University 

BRADY,  SCOTT,  T.,  Case  Western  Reserve  University 

BREITWIESER,  GERDA  E.,  Washington  University  Medical  School 

BRINLEY,  S.  J.,  National  Institutes  of  Health 

BROUSSEAU,  DIANE  J.,  Fairfield  University 

BUCHANAN,  Jo  ANN,  Northeastern  University 

BURGESS,  BARBARA  K.,  Charles  F.  Kettering  Research  Laboratory 

BURSZTAJN,  SHERRY,  Baylor  College  of  Medicine 

CAPLOW,  MICHAEL,  University  of  North  Carolina 

CARIELLO,  Lucio,  Stazione  Zoologica,  Italy 

CARLTON,  DEBORAH  A.,  Woods  Hole  Oceanographic  Institution 

CARTER,  JACQUELINE  M.,  Hunter  College 

CHRISTAKIS,  NICHOLAS,  Washington,  DC 

CLAPIN,  DAVID  F.,  University  of  Ottawa,  Canada 

CLARK,  JOHN  M.,  Michigan  State  University 

COHEN,  JOY,  New  Orleans,  LA 

COHEN,  JERRY,  Johns  Hopkins  University 

COHEN,  ROCHELLE  S.,  University  of  Illinois  College  of  Medicine 

COLLINS,  STEPHEN,  Case  Western  University 

COMOGLIO,  PAOLO,  University  of  Torino,  Italy 

CONWAY,  KEVIN,  Johns  Hopkins  University 

CORNETT,  ROBERT,  Iowa  State  University 

CROWTHER,  BOB,  Wistar  Institute 

CZETO,  ALEXANDER,  Mellon  Institute 

CZINN,  STEVEN  J.,  Rainbow  Babies  and  Children's  Hospital 

DENNISON,  WILLIAM,  University  of  Chicago 

DETOLEDO-MORRELL,  LEYLA,  Rush  Medical  Center 

DICKER,  ADAM,  Columbia  College 

DiPoLO,  REINALDO  V.,  IVIC,  Venezuala 

DIXON,  ANDREW,  Case  Western  Reserve  University  Medical  School 

DONOHUE,  MELANIE,  Boston  University 

DUNHAM,  PHILIP,  Syracuse  University 

ECKBERG,  WILLIAM  R.,  Howard  University 

ECKERT,  RICHARD,  Lehman  College,  CUNY 

EHRENSTEIN,  GERALD,  National  Institutes  of  Health 

EHRING,  GEORGE  R.,  University  of  California,  San  Francisco 

EHRLICH,  BARBARA,  Albert  Einstein  College  of  Medicine 

EISEN,  ANDREW,  University  of  Pennsylvania 

EISEN,  MATTHEW,  Harvard  University 

ELLNER,  JERROLD,  University  Hospitals  of  Cleveland 

PATH,  KARL  R.,  Case  Western  Reserve  University 

FENN,  JANE,  University  of  Texas  Medical  Branch 


88  MARINE  BIOLOGICAL  LABORATORY 

FERNANDEZ,  JULIO  M.,  University  of  California 

FOHLMEISTER,  JuRGEN  F.,  University  of  Minnesota 

FOLLEY,  LINDA  S.,  Brown  University 

FORSCHER,  PAUL,  University  of  North  Carolina 

FRACE,  ALAN  M.,  University  of  Texas  Medical  Branch 

FRENCH,  KATHLEEN,  University  of  North  Carolina  at  Chapel  Hill 

FRIDOVICH,  JUDITH  L.,  Princeton  University 

GAINER,  HAROLD,  National  Institutes  of  Health 

GALLANT,  PAUL,  National  Institutes  of  Health 

GART,  SERGE,  Marlboro  College 

GEDULDIG,  ULLA,  St.  John's,  Canada 

GILLY,  WILLIAM  F.,  University  of  Pennsylvania 

GIUDITTA,  ANTONIO,  International  Institute  of  Genetics  and  Biophysics,  Italy 

GOULD,  ROBERT,  New  York  Institute  for  Basic  Research  in  Mental  Health 

GRUPP,  STEPHEN,  Cincinnati,  OH 

GRZYWACZ,  NORBERTO,  Hebrew  University  of  Jerusalem 

GUCHARDI,  JOHN,  Scarborough  College  University  of  Toronto,  Canada 

GUEVARA,  MICHAEL  R.,  McGill  University 

GUTERMAN,  LEE,  Binghamton,  NY 

GUTSTEIN,  DAVID,  Hamilton  College 

HAGELSTEIN,  ERIC  B.,  Northwestern  University  Medical  School 

HALL,  SHERWOOD,  IWS  Seward  Marine  Station,  Alaska 

HALVORSEN,  LISA,  Vassar  College 

HANKIN,  MARK  H.,  Case  Western  Reserve  University 

HARRIS,  ANDREW  L.,  Albert  Einstein  College  of  Medicine 

HARRIS,  EDWARD  M.,  Duke  University  Medical  Center 

HAYASHI,  JOHN,  University  of  North  Carolina 

HAYS,  TOM,  University  of  North  Carolina 

HEMPEL,  BILL,  Pomona  College 

HERLANDS,  Louis,  Rockefeller  University 

HINES,  MICHAEL,  Duke  University  Medical  Center 

HINOJOSA,  J.  U.,  Texas  Medical  Branch 

HONKANEN,  ANITA,  Massachusetts  Institute  of  Technology 

HOSHI,  TOSHINORI,  University  of  New  Hampshire 

HYLAND,  ANAISA  D.,  City  College  of  CUNY 

IANNACCONE,  VICTOR,  Rutgers  State  University 

INOMATA,  HACHIRO,  Tohoku  University  School  of  Medicine,  Japan 

INOUE,  HIDEYO,  Mellon  Institute 

IWASA,  KUNIHIKO,  National  Institutes  of  Health 

JACOBSEN,  FREDA,  University  of  Cincinnati 

JASLOVE,  STEWART,  Albert  Einstein  College  of  Medicine 

JOHNSON,  THOMAS,  Case  Western  Reserve  University 

JOHNSON,  WARREN,  Amray  Inc. 

KACHAR,  BECHARA,  National  Institutes  of  Health 

KAHLER,  STEPHEN,  Emory  University  School  of  Medicine 

KANDEL,  PAUL,  Haverford  College 

KAO,  PETER  N.,  Columbia  University  College  of  Physicians  and  Surgeons 

KAPLAN,  EHUD,  Rockefeller  University 

KAPUR,  RAJ  P.,  University  of  California,  Los  Angeles 

KASS,  LEONARD,  Syracuse  University 

KATZ,  BARRY  R.,  Case  Western  Reserve  University 

KATZ,  MICHAEL,  Brown  University 

KAUPP,  U.  BENJAMIN,  SUNY  at  Stony  Brook 

KAWAI,  MASATAKA,  Columbia  University 

KELLER,  RAYMOND  E.,  University  of  California 

KEYNAN,  ALEX,  Hebrew  University,  Israel 


RESEARCH  AND  TRAINING  PROGRAMS  89 

KIEHART,  DANIEL  P.,  Johns  Hopkins  University  School  of  Medicine 

KIRCHMAN,  DAVID,  Harvard  University 

KOIDE,  S.  S.,  Rockefeller  University 

KOMM,  BARRY,  University  of  South  Florida,  Tampa 

KRACKE,  GEORGE  R.,  Washington  University  School  of  Medicine 

K.RAUTHAMER,  VICTOR,  New  York  Medical  College 

LAFORET,  GENEVIEVE,  Chestnut  Hill,  MA 

LANGAGER,  JANIS,  Biozentrum,  Basel,  Switzerland 

LEIGHTON,  STEPHEN  B.,  National  Institutes  of  Health 

LESLIE,  ROGER,  Boulder,  CO 

LEUCHTAG,  H.  RICHARD,  University  of  Texas  Medical  Branch 

LEWENSTEIN,  LISA,  New  York  Medical  College 

LINFANG,  WANG,  Rockefeller  University 

LIPETZ,  LEO  E.,  Ohio  State  University 

LIPMAN,  DEBORAH,  National  Resource  Defense  Council 

LLANO,  MARIA  I.,  University  of  California 

Lo,  Woo-KuEN,  Kresge  Eye  Institute 

Lo  BUE,  CHARLES,  Albert  Einstein  College  of  Medicine 

LOPEZ-BARNEO,  JOSE,  University  of  Pennsylvania 

Luzzi,  LYNN,  Iowa  State  University 

LYN-COOK,  LASCELLES  E.,  University  of  North  Carolina 

LYTTLE,  C.  RICHARD,  University  of  Pennsylvania 

MACHIDA,  KOICHI,  University  of  Miami  School  of  Medicine 

MALEMUD,  CHARLES  J.,  Case  Western  Reserve  University 

MANCINI,  VIVIAN,  Hunter  College 

MASTROIANNI,  LUIGI,  University  of  Pennsylvania 

MATHEWS,  RITA  W.,  Hunter  College 

MATTESON,  RICHARD,  University  of  Pennsylvania 

MCCARTHY,  ROBERT,  Dartmouth  College 

MCKINNEY,  LESLIE  C.,  Washington  University 

MEEDEL,  THOMAS  H.,  Wistar  Institute 

MEINERTZHAGEN,  I.  A.,  Dalhousie  University 

MISEVIC,  GRADIMIR,  University  of  Basel,  Switzerland 

MOON,  RANDALL  T.,  University  of  Washington 

MORAN,  MICHAEL  N..,  Emory  University 

MORAN,  WILLIAM  M.,  University  of  Maryland 

MOREAU,  MARC,  Station  Biologique,  France 

MORRIS,  JAMES  R.,  Case  Western  Reserve  University 

MURAMATSU,  IKUNOBU,  Northwestern  University  Medical  School 

MURPHY,  RODNEY,  SUNY  at  Albany 

NORTHCUTT,  MARY  SUE,  University  of  Michigan 

OBAID,  ANALIA,  University  of  Miami  School  of  Medicine 

OBARA,  SHOSAKU,  Albert  Einstein  College  of  Medicine 

OFFNER,  GWYNNETH,  Boston  University  School  of  Medicine 

ORBACH,  HARRY,  Yale  University 

PABORSKY,  LISA,  Laboratory  of  Biophysics,  MBL 

PALTI,  YORAM,  Technical  Medical  School,  Israel 

PANT,  HARISH  C.,  National  Institute  on  Alcohol  Abuse  and  Alcoholism 

PAXHIA,  TERESA  M.,  University  of  Rochester 

PEARCE,  JOANNE  M.,  Scarborough  College,  Canada 

PERSELL,  ROGER,  Mercy  College 

PETHIG,  RONALD,  University  College  of  North  Wales,  Great  Britain 

POUSSART,  DENIS,  Laval  University,  Canada 

POWERS,  MAUREEN  K.,  Vanderbilt  University 

PRUSCH,  ROBERT  D.,  Gonzaga  University 

QUINTA-FERREIRA,  EMILIA,  University  of  East  Anglia,  England 


90  MARINE  BIOLOGICAL  LABORATORY 

RABIN,  DANIEL,  Biozentrum,  University  of  Basel,  Switzerland 

RADU,  AURELIAN,  University  of  Miami  Medical  School 

RAKOWSKI,  ROBERT  F.,  Washington  University  School  of  Medicine 

RAM,  JEFFREY,  Wayne  State  University  School  of  Medicine 

RAPPORT,  SETH,  Hudson,  NY 

RASMUSSEN,  HOWARD,  Yale  University  School  of  Medicine 

REDDY,  VINAY  N.,  Wayne  State  University  School  of  Medicine 

RICHERSON,  GEORGE,  University  of  Iowa 

RICKARD,  CHARLES  G.,  Cornell  University 

RIEMANN,  Bo,  University  of  Copenhagen 

RILEY,  WILLIAM,  Amray  Inc. 

ROBERTSON,  LOLA  E.,  American  Museum  of  Natural  History 

ROSE,  BIRGIT,  University  of  Miami  School  of  Medicine 

ROSENTHAL,  ERIC,  Harvard  Medical  School 

ROTH,  VICTORIA,  Mount  Sinai  Hospital 

ROUTZAHN,  JOHN  A.,  National  Institutes  of  Health 

RUSHBROOK,  JULIE  I.,  SUNY,  Downstate  Medical  Center 

SALGADO,  VINCENT  L.,  University  of  California 

SANGER,  TERRY,  New  York  City,  NY 

SAPIRO,  JACOB,  Case  Western  Reserve  University 

SAPIRO,  KATHERINE,  Case  Western  Reserve  University 

SARVET,  NANCY,  New  York,  NY 

SAUNDERS,  BARRY,  Chapel  Hill,  NC 

SCHATZ,  SCOTT,  University  of  Massachusetts,  Amherst 

SCHLUMPBERGER,  JAY  M.,  Stanford  University 

SCHLUP,  VERENA,  Biozentrum,  University  of  Basel,  Switzerland 

SCHWARTZ,  JAMES  H.,  Columbia  University 

SCRUGGS,  VIRGINIA  M.,  Northwestern  University  Medical  School 

SELMAN,  KELLY,  University  of  Florida  School  of  Medicine 

SENSEMAN,  DAVID,  Monell  Chemical  Senses  Center 

SERHAN,  CHARLES,  New  York  University  Medical  Center 

SHOAF,  SARA,  Johns  Hopkins  University 

SHRIER,  ALVIN,  McGill  University,  Canada 

SHRIVASTAV,  BRIJ  B.,  Duke  University  Medical  Center 

SIMON,  SANFORD,  New  York  University  Medical  Center 

SIMONNEAU,  MICHEL,  Laboratoire  de  Neurobiologie  Cellulaire,  France 

SINGH,  H.  B.,  University  of  Toronto,  Canada 

SMITH,  LAURENS  H.,  University  of  Maryland 

Socci,  ROBIN  R.,  Rutgers  University 

SPRAY,  DAVID  C.,  Albert  Einstein  College  of  Medicine 

STEINACKER,  ANTOINETTE,  Rockefeller  University 

STERN,  JEFFREY  H.,  Brandeis  University 

STOPAK,  SAMUEL,  Atlanta,  GA 

STRACHER,  ALFRED,  SUNY,  Downstate  Medical  Center  in  Brooklyn 

SUGIMORI,  MUTSUYUKI,  New  York  University  Medical  Center 

SUPRENANT,  KATHY,  University  of  Virginia 

SUSAN,  STANLEY,  Wayne  State  University 

SWENSON,  RANDOLPHE,  University  of  Pennsylvania 

SZARO,  BEN,  Johns  Hopkins  University 

SZENTKIRALYI-SZENT-GYORGYI,  EVA  M.,  Brandeis  University 

TAATJES,  DOUGLAS,  Kansas  State  University 

TANSEY,  TERESE,  Harvard  Medical  School 

TIFFERT,  TERESA,  University  of  Maryland 

TOMPKINS,  ROBERT,  Tulane  University 

TRAEGER,  EVELINE  C.,  SUNY  at  Buffalo 

TRINKAUS-RANDALL,  VICKERY,  University  of  Wisconsin 


RESEARCH  AND  TRAINING  PROGRAMS  91 

VARMA,  VIVEK,  Howard  University 

VASSORT,  GUY,  CNRS,  University  of  Paris,  France 

VERAKALASA,  PACHARA,  University  of  Hawaii 

VIERLING,  ELIZABETH,  University  of  Chicago 

WALRATH,  DANA,  Columbia  University 

WANG,  HOWARD  H.,  University  of  California 

WEISS,  JERRY  S.,  Northwestern  University 

WEISSMAN,  IRVING,  Stanford  University  School  of  Medicine 

WELLS,  DAN  E.,  Indiana  University 

WESTERFIELD,  MONTE,  University  of  Oregon 

WESTERMAN,  LARRY,  Syracuse  University 

WHITE,  MICHAEL  M.,  Brandeis  University 

WHITE,  ROY  LEE,  National  Institutes  of  Health 

WHITEHEAD,  DENEENE,  Hunter  College 

WHITTEMBURY,  JOSE,  Universidad  Peruana  Cayetano  Heredia,  Peru 

WICKS,  GEORGE  E.,  University  of  Baltimore 

WILLIAMS,  HARRIET  D.,  Yale  University 

WORTHINGTON,  ALMA,  Carnegie-Mellon  University 

Wu,  CHAU  H.,  Northwestern  University 

YEH,  JAY  Z.,  Northwestern  University 

YULO,  TERESA,  University  of  Rochester  School  of  Medicine 

ZAKEVICIUS,  JANE,  New  York  University  School  of  Medicine 

ZEBLEY,  ELMER,  New  College  of  the  University  of  South  Florida 

ZIMERING,  MARK  B.,  Albert  Einstein  College  of  Medicine 

ZIMMERMAN,  ANITA,  University  of  Miami  School  of  Medicine 

ZIMMERMAN,  MORRIS,  Merck  Institution  for  Therapeutic  Research 

YEAR-ROUND  PROGRAMS 

BOSTON  UNIVERSITY  MARINE  PROGRAM  (BUMP) 

Director 

WHITTAKER,  J.  RICHARD,  Boston  University/Marine  Biological  Laboratory 

Staff  (of  Boston  University  unless  otherwise  indicated) 

ALLEN,  SARAH 

ATEMA,  JELLE 

CROWTHER,  ROBERT 

GOVIND,  C.  K.,  University  of  Toronto 

HAHN,  DOROTHY 

HARTMAN,  JEAN,  University  of  Connecticut 

HILL,  RUSSELL,  University  of  Toronto 

HUMES,  ARTHUR 

LOESCHER,  JANE 

MEEDEL,  THOMAS 

PRICE,  CHRISTOPHER 

RAYCROFT,  KATHLEEN 

TAMM,  SIDNEY 

TAMM,  SIGNHILD 

TAYLOR,  MARGERY 

VALIELA,  IVAN 

VAN  ETTEN,  RICHARD 

Students  (of  Boston  University  unless  otherwise  indicated) 

BARSHAW,  DIANA  BRYANT,  DONALD 

BRYANT,  BRUCE  BUCHSBAUM,  ROBERT 


92 


MARINE  BIOLOGICAL  LABORATORY 


CARACO,  NINA 

CLARKE,  JOANN 

COHEN,  ROSALINE,  National  Marine 

Fisheries  Service 
COSTA,  JOSEPH 
DAVIS,  CABELL 
DOJIRI,  MASAHIRO 
FERME,  PAOLA 
FOREMAN,  KENNETH 
FUJITA,  RODNEY 
GODDARD,  KATHRYN 


HALL,  VALERIE 
HOWES,  BRIAN 
JOHNSON,  BRUCE 
MACIOLEK-BLAKE,  NANCY 
Moss,  ANTHONY 
PASCOE,  NATALIE 
POOLE,  ALAN 

RlTTENHOUSE,  ANN 

TROTT,  THOMAS 
WILLIAMS,  ISABELLE 
WILSON,  JOHN 


DEVELOPMENTAL  AND  REPRODUCTIVE  BIOLOGY  LABORATORY 

Director 

GROSS,  PAUL  R.,  Marine  Biological  Laboratory 

Staff  (of  Marine  Biological  Laboratory  unless  otherwise  indicated) 

CARIELLO,  Lucio,  Stazione  Zoologica,  Naples,  Italy 

O'LouGHLiN,  JOHN 

SIMPSON,  ROBERT  T.,  National  Institutes  of  Health 


THE  ECOSYSTEMS  CENTER 

Director 

WOODWELL,  GEORGE  M.,  Marine  Biological  Laboratory 


Staff  and  consultants  (all  of  Marine  Biological  Laboratory) 


BADENHAUSEN,  MARGUERITE  M. 

BARKLEY,  JOHN 

BANNER,  STEVEN 

BEALE,  ELEANORE  M. 

BEARD,  SARAH  H. 

BOWLES,  FRANCIS  P. 

BURROUGHS,  RICHARD  H. 

CARLSON,  CHRISTOPHER 

CHAN,  Yip-Hoi 

COLE,  JONATHAN 

CORLISS,  TERESA  A.  L. 

DUNCAN,  THOMAS 

DUNGAN,  JENNIFER 

ELDRED,  KATE 

ELKIN,  KERRY 


FOWNES,  JAMES  N. 
GARRITT,  ROBERT  H. 
GIBBS,  RICHARD  K. 
GREGG,  DAVID 
GUTJAHR,  RUTH  E. 
HELFRICH,  JOHN  V.  K. 
HOBBIE,  JOHN  E. 
HOUGHTON,  RICHARD,  A. 
HOWARTH,  ROBERT  W. 
JUERS,  DAVID  W. 
KANE,  ANN  E. 

KlJOWSKI,  VOYTEK 

LAJTHA,  KATHRYN 
MARINO,  ROXANNE 
MARINUCCI,  ANDREW  C. 


RESEARCH  AND  TRAINING  PROGRAMS  93 

MARQUIS,  SALLY  L. 

MELILLO,  JERRY  M.  SCHIMEL,  JOSHUA 

MILLINGER,  MYRA  SECHOKA,  ELIZABETH  M. 

MONTGOMERY,  ELLYN  SEMINO,  SUZANNE 

MONTGOMERY,  MARY  LOUISE  SHAVER,  GAIUS  R. 

MORRIS,  JAMES  T.  SHAW,  JOAN 

Moss,  ANN  H.  SIMMONS,  NANCY  S. 

PALM,  CHERYL  A.  SLADOVICH,  HEDY  E. 

PARSONS,  KATHERINE  C.  STEUDLER,  PAUL  A. 

PETERSON,  BRUCE  J.  TURNER,  ANDREA  R. 

QUICK,  DEBORAH  G.  UPTON,  JOAN  M. 


Trainees 

BOWDEN,  WILLIAM  B.,  North  Carolina  State  University,  Year-in-Science 
CAVANAUGH,  COLLEEN,  Harvard  University,  Year-in-Science 
DAUKAS,  PAULA,  Yale  University,  Year-in-Science 
DICK,  RANDALL  W.,  Intern 
GORDON,  DORIA,  Intern 
LANG,  HELEN,  Intern 
NEWHART,  GARY,  Intern 
WILLEY,  JOANNE,  Intern 


LABORATORY  OF  BIOPHYSICS 

Director 

ADELMAN,  WILLIAM  J.,  JR.,  NINCDS-NIH 

Staff  (of  NINCDS-NIH  unless  otherwise  indicated) 

Section  on  Neural  Membranes 

ADELMAN,  WILLIAM  J.,  JR.,  Chief 

BROWN,  DAVID 

CLAY,  JOHN  R. 

DEFELICE,  Louis  J. 

DYRO,  FRANCES  M.,  Veterans  Administration  Medical  Center 

GOLDMAN,  DAVID  E.,  State  University  of  New  York  at  Binghamton 

HODGE,  ALAN  J. 

LAFORET,  GENEVIEVE 

LEONARD,  DOROTHY  A. 

MUELLER,  RUTHANNE 

RICE,  ROBERT  V.,  Carnegie-Mellon  University 

ROSLANSKY,  PRISCILLA  F.,  Bunting  Institute  of  Radcliffe  College 

RYAN,  LIANE  E. 

SHIMAN,  LEON  G. 

SHOUKIMAS,  JONATHAN  J. 

TYNDALE,  CLYDE  L. 

WALTZ,  RICHARD  B. 

WELLS,  JAY  B. 


94  MARINE  BIOLOGICAL  LABORATORY 

Section  on  Neural  Systems 

ALKON,  DANIEL  L.,  Chief 

ACOSTA-URQUIDI,  JUAN 

BUCHANAN,  JoANN,  Northeastern  University 

FARLEY,  JOSEPH,  Princeton  University 

GART,  SERGE,  University  of  Vermont 

GOH,  YASUMASA 

HARRIGAN,  JUNE  F. 

HILL,  LENA 

KUZIRIAN,  ALAN  M. 

KUZIRIAN,  JEANNE 

LEDERHENDLER,  IZJA 

LEIGHTON,  STEPHEN 

NEARY,  JOSEPH  T. 

RAM,  JEFFREY,  Wayne  State  University 

RICHARDS,  WILLIAM,  Princeton  University 

SENFT,  STEPHEN  L.,  Washington  University 


LABORATORY  FOR  MARINE  ANIMAL  HEALTH 

Director 

LEIBOVITZ,  Louis,  New  York  State  College  of  Veterinary  Medicine 

Staff 

ABT,  DONALD  A.,  University  of  Pennsylvania 
RICKARD,  CHARLES  C.,  Cornell  University 
STONE,  AMY,  Cornell  University 

LABORATORY  OF  SENSORY  PHYSIOLOGY 

Director 

MACNICHOL,  EDWARD  F.,  JR.,  Marine  Biological  Laboratory 

Staff  (all  of  Marine  Biological  Laboratory) 

COLLINS,  BARBARA  ANN 
COOK,  PATRICIA  B. 
CORSON,  D.  WESLEY 
FEIN,  ALAN 
HAROSI,  FERENC  I. 
LEVINE,  JOSEPH  S. 
LEVY,  SIMON 
PAYNE,  RICHARD 
SZUTS,  ETE  ZOLTAN 

NATIONAL  FOUNDATION  FOR  CANCER  RESEARCH 
Director 
SZENT-GYORGYI,  ALBERT,  Marine  Biological  Laboratory 


RESEARCH  AND  TRAINING  PROGRAMS  95 

Staff  (of  Marine  Biological  Laboratory  unless  otherwise  indicated) 

GASCOYNE,  PETER  R.  C. 

MCLAUGHLIN,  JANE  A. 

MEANY,  RICHARD  A. 

PETHIG,  RONALD,  University  College  of  North  Wales,  United  Kingdom 


LABORATORY  OF  CARL  J.  BERG,  JR. 

Director 

BERG,  CARL  J.,  JR.,  Marine  Biological  Laboratory 

Staff  (of  Marine  Biological  Laboratory  unless  otherwise  indicated) 

ADAMS,  NANCY 

ALATALO,  PHILIP 

BROUSSEAU,  DIANE,  Fairfield  University 

DAVIS,  JONATHAN,  Yale  University 

EARLY,  JULIE 

TURNER,  RUTH  D.,  Harvard  University 


LABORATORY  OF  D.  EUGENE  COPELAND 

Director 

COPELAND,  D.  EUGENE,  Marine  Biological  Laboratory 


LABORATORY  OF  JUDITH  P.  GRASSLE 

Director 

GRASSLE,  JUDITH  P.,  Marine  Biological  Laboratory 

Staff  (of  Marine  Biological  Laboratory  unless  otherwise  indicated) 

HILL,  SUSAN  DOUGLAS,  Michigan  State  University 
MILLS,  SUSAN 
SCHOTT,  EDWARD 

LABORATORY  OF  SHINYA  INOUE 

Director 

INOUE,  SHINYA,  University  of  Pennsylvania/Marine  Biological  Laboratory 

Staff  (of  the  Marine  Biological  Laboratory  unless  otherwise  indicated) 

EISEN,  ANDREW,  University  of  Pennsylvania 

INOUE,  CHRISTOPHER 

INOUE,  THEODORE 

LUTZ,  DOUGLAS,  University  of  Pennsylvania 

WOODWARD,  BERTHA  M. 


96  MARINE  BIOLOGICAL  LABORATORY 


Visiting/Col laboratory  Investigators 

TANAKA,  YUICHIRO,  Sugashima  Marine  Biological  Station,  Japan 
TILNEY,  LEWIS  G.,  University  of  Pennsylvania 
WOODRUFF,  RICHARD  I.,  West  Chester  State  College 


LABORATORY  OF  ERIC  KANDEL 

Director 

KANDEL,  ERIC,  Columbia  University 

Staff 

CAPO,  THOMAS,  Columbia  University 
PAIGE,  JOHN  A.,  Columbia  University 
PERRITT,  SUSAN,  Columbia  University 

LABORATORY  OF  JEFFRY  B.  MITTON 

Director 

MITTON,  JEFFRY  B.,  University  of  Colorado 

Staff 

CARLTON,  DEBORAH,  University  of  California  at  Davis 

LABORATORY  OF  CAROL  L.  REINISCH 

Director 

REINISCH,  CAROL  L.,  Tufts  University  School  of  Veterinary  Medicine 

Staff 

CHARLES,  ANN  M.,  Tufts  University  School  of  Veterinary  Medicine 


LABORATORY  OF  OSAMU  SHIMOMURA 

Director 

SHIMOMURA,  OSAMU,  Princeton  University 

Staff 

SHIMOMURA,  AKEMI,  Princeton  University 

LABORATORY  OF  RAYMOND  E.  STEPHENS 

Director 

STEPHENS,  RAYMOND  E.,  Marine  Biological  Laboratory /Boston  University  Medical  School 


RESEARCH  AND  TRAINING  PROGRAMS  97 


Staff 


PORTER,  MARY  E.,  Marine  Biological  Laboratory/University  of  Pennsylvania 
PRATT,  MELANIE,  Harvard  Medical  School 

STOMMEL,  ELIJAH,  Marine  Biological  Laboratory/Boston  University  Medical  School 
SUPRENANT,  KATHY,  University  of  Virginia 

LABORATORY  OF  J.  RICHARD  WHITTAKER 

Director 

WHITTAKER,  J.  RICHARD,  Boston  University/Marine  Biological  Laboratory 

Staff  {of  Boston  University  unless  otherwise  indicated) 

CROWTHER,  ROBERT 

LOESCHER,  JANE  L. 

MEEDEL,  THOMAS  H. 

Wu,  S.  C.,  visiting  investigator,  Academia  Sinica,  People's  Republic  of  China 

XII.  HONORS 
FRIDAY  EVENING  LECTURES 

GERHARDT,  CARL,  University  of  Missouri  at  Columbia,  January  9,  "Sound  Pattern  Rec- 
ognition in  North  American  Tree  Frogs:  Neurobiological  Implications'" 

RASMUSSEN,  HOWARD,  Yale  University,  January  16,  "Calcium  and  Cyclic- AMP  as  Syn- 
archic  Messengers" 

LIEM,  KAREL,  Harvard  University,  January  23,  "Functional  Morphology  of  the  Feeding 
Apparatus  of  Fishes:  Do  Fish  Defy  Gauss'  Principle?" 

DARNELL,  JAMES  E.  Rockefeller  University,  June  26,  "Consideration  of  Animal  Cell  Func- 
tion and  Evolution" 

HOBBIE,  JOHN  E.,  Marine  Biological  Laboratory,  July  3,  "Process  Regulation  in  an  Arctic 
Ecosystem" 

HEUSER,  JOHN,  Washington  University  School  of  Medicine,  July  9,  10,  Forbes  Lectures.  I. 
"Structural  Basis  of  Synoptic  Transmission"  II.  "A  3-D  Journey  Through  the  Interior 
of  Nerve  and  Muscle  Cells" 

NICHOLLS,  JOHN  G.,  Stanford  University,  July  17,  Lang  Lecture,  "One  Cell  at  a  Time:  The 
Analysis  of  a  Simple  Nervous  System" 

STEITZ,  JOAN  A.,  Yale  University,  July  24,  "Autoantibodies  as  Probes  for  Small  Ribonu- 
cleoproteins  from  Eukaryotes" 

MclNTOSH,  J.  RICHARD,  University  of  Colorado,  Boulder,  July  31,  "Mitotic  Mechanism: 
Ever  Interesting,  Still  Elusive" 

SAGER,  RUTH,  Sidney  Farber  Cancer  Institute,  August  7,  "DNA  Methylation:  From 
Chlamydomonas  to  Cancer" 

INOUE,  SHINYA,  Marine  Biological  Laboratory,  August  14,  "Form,  Movement,  and  Life: 
Adventures  in  Light  Microscopy" 

GROSS,  JEROME,  Massachusetts  General  Hospital,  August  21,  Zwilling  Lecture,  "Regulation 
of  Collagenase  by  Cell -Cell  Interactions" 

SOMERO,  GEORGE  N.,  Scripps  Institution  of  Oceanography,  August  28,  "Protein  Adaptation 
to  the  Physical  Environment:  Discerning  Basic  Molecular  'Themes'  Through  the  Study 
of  Their  'Variations' ' 

CHARLES  A.  LINDBERGH  LECTURES  IN  ECOLOGY 

MANN,  KENNETH  H.,  Bedford  Institute  of  Oceanography,  June  24,  "Management  of  Re- 
sources in  the  Coastal  Zone:  Laminaria  and  Lobsters  in  Nova  Scotia" 


98  MARINE  BIOLOGICAL  LABORATORY 

LOVEJOY,  THOMAS  E.,  World  Wildlife  Fund,  U.  S.,  July  8,  "Conserving  Wildlife  in  a  Frag- 
mented World"" 

BOLIN,  BERT,  University  of  Stockholm,  July  29,  "Man's  Interference  with  the  Biosphere  on 
a  Global  Scale" 

ASSOCIATES'  LECTURE 

MARGULIS,  LYNN,  Boston  University,  August  1,  "The  Earliest  Life  on  Earth" 

» 

SPECIAL  LECTURE 

HORRIDGE,  G.  ADRIAN,  Australian  National  University,  July  12,  "New  Work  on  the  Insect 
Compound  Eye" 

ROCKEFELLER  FOUNDATION  LECTURE  SERIES  "UNDERSTANDING  SCIENTIFIC 
INFORMATION  SYSTEMS  AND  OPTIMIZING  INFORMATION  RETRIEVAL" 

GOFFMAN,  WILLIAM,  Case  Western  Reserve  University,  July   13,  "The  Ecology  of  the 

Biomedical  Literature" 
WARREN,  KENNETH  S.,  Rockefeller  Foundation,  July  14,  "The  Quantitative  and  Qualitative 

Structures  of  the  Biomedical  Literature" 
MOSTELLER,  FREDERICK,  Harvard  School  of  Public  Health,  July  15,  "Design  and  Evaluation 

of  Biomedical  Studies" 
GOFFMAN,  WILLIAM,  Case  Western  Reserve  University,  July  16,  "Information  Retrieval 

Strategies" 
GARFIELD,  EUGENE,  Institute  for  Scientific  Information,  July  17,  "Information  Retrieval 

Systems" 

GRASS  FOUNDATION  FELLOWS 

BARISH,  MICHAEL  E.,  University  of  California 

BODZNICK,  DAVID,  Wesleyan  University,  Associate  Program  Director 

BRUNKEN,  WILLIAM  J.,  New  York  University  Medical  Center 

CHAD,  JOHN  E.,  University  of  California 

DENKIN,  MICHAEL  S.,  SUNY  at  Albany 

FERNANDEZ,  JULIO,  University  of  California  at  Los  Angeles 

FRESCHI,  JOSEPH  E.,  AFRRI 

FROHLICH,  AMALIE,  Dalhousie  University 

GLANZMAN,  DAVID  L.,  University  of  California 

GRAF,  WERNER  M.,  New  York  University  Medical  Center 

JAMES-KRACKE,  MARILYN,  Washington  University 

KILDUFF,  THOMAS  S.,  Stanford  University 

LLANO,  ISABEL,  University  of  California  at  Los  Angeles 

McKiNNEY,  LESLIE  C.,  Washington  University 

REUBEN,  JOHN  P.,  Columbia  University,  Program  Director 

VANDENBERG,  CAROL  A.,  University  of  California 

JOSIAH  MACY,  JR.,  FOUNDATION  SCHOLARS 

BAKER,  TAHIRIH,  Jackson  State  University 
BELL,  BARBARA  JEAN,  Atlanta  University 
BOLDEN,  MARSHA,  Texas  Southern  University 
BROWN,  JANICE,  Tougaloo  College 
COLEMAN,  EDWARD,  Texas  Southern  University 
DUROJAIYE,  MUSTAPHA,  Atlanta  University 
ELLIOTT,  WANDA,  Jackson  State  University 


HONORS  99 

FLOYD,  CARL,  Morehouse  College 

FLOYD,  PATRICIA,  Morehouse  College 

GREEN,  KAREN,  Dillard  University 

HUBBARD,  KAREN,  Illinois  Institute  of  Technology 

JENKINS,  GAYE,  Dillard  University 

JOHNSON,  DENISE,  Texas  Southern  University 

JORDAN,  THOMAS,  Dillard  University 

NORMAN,  PHILIPPA,  Tougaloo  College 

PETTIS,  RENEE,  Texas  Southern  University 

SANDERS,  PAMELA,  Texas  Southern  University 

SMITH,  MARVA,  Dillard  University 

SPEARS,  CLIFTON,  JR.,  Dillard  University 

VERRETT,  JOYCE,  Dillard  University 

WALKER,  ROSIE,  Tougaloo  College 

STEPS  TOWARD  INDEPENDENCE  FELLOWS 

BEGG,  DAVID,  Harvard  Medical  School 

BELL,  WAYNE,  Hamilton  College 

BOYER,  BARBARA,  Union  College 

BRENCHLEY,  GAYLE,  University  of  California  at  Irvine 

BULLOCK,  JAMES,  Rush  University 

CHARLTON,  MILTON,  Ohio  University  College  of  Medicine 

HAIMO,  LEAH,  University  of  California  at  Riverside 

HILL,  SUSAN,  Michigan  State  University 

KIRSCH,  GLENN,  Rutgers  University 

KOEHL,  MIMI,  University  of  California  at  Berkeley 

SALAMA,  GUY,  University  of  Pittsburgh  School  of  Medicine 

SMITH,  STEPHEN,  Yale  University 

TELZER,  BRUCE,  Pomona  College 

TYTELL,  MICHAEL,  Bowman  Gray  School  of  Medicine 

WATSON,  WINSOR,  University  of  New  Hampshire 

GARY  N.  CALKINS  MEMORIAL  SCHOLARSHIP 
DON  CARLOS,  LYDIA,  Northeastern  Ohio  Universities  College  of  Medicine 

FRANCES  S.  CLAFF  MEMORIAL  SCHOLARSHIP 
JUNG,  LADONNA,  Columbia  University 

EDWIN  GRANT  CONKLIN  MEMORIAL  SCHOLARSHIP 

KEEN,  SUSAN,  University  of  Michigan 
PALLAS,  SARAH,  Cornell  University 

LUCRETIA  CROCKER  SCHOLARSHIPS 

BRODFEUHRER,  PETER,  University  of  Virginia 

DAVIS,  JONATHAN,  Yale  University 

HALS,  GARY,  Capital  University 

MOORE,  DARRELL,  University  of  Texas  at  Austin 

SHAMMA,  SHIHAB,  Stanford  University 


100  MARINE  BIOLOGICAL  LABORATORY 

FOUNDERS  SCHOLARSHIPS 

In  1981,  these  Scholarships  were  given  in  memory  of: 

W.  C.  CURTIS 
CASWELL  GRAVE 
L.  V.  HEILBRUNN 
OTTO  LOEWI 
S.  O.  MAST 
T.  H.  MORGAN 
A.  H.  STURTEVANT 

Recipients: 

CHOU,  YING-HAO,  University  of  Virginia 

DUNN-COLEMAN,  ELAINE,  University  of  Virginia 

GRIMWADE,  BRIAN,  Yale  University 

KOBAYASHI,  YOSHITERU,  University  of  Tokyo,  Japan 

LUTZ,  DOUGLAS,  University  of  Pennsylvania 

OLIVERIRA,  ANA,  Universidade  Federal  Do  Rio  De  Janeiro,  Brazil 

SPAIN,  LISA,  Indiana  University 

WAGNER,  JEFFERY,  State  University  of  New  York  at  Buffalo 

WARD,  GARY,  University  of  California  at  San  Diego 

ALINE  D.  GROSS  SCHOLARSHIP 

KEEN,  SUSAN,  University  of  Michigan 

OLIVERIRA,  ANA,  Universidade  Federal  Do  Rio  De  Janeiro,  Brazil 

MERKEL  H.  JACOBS  SCHOLARSHIP 
NOVICKI,  ANDREA,  University  of  Hawaii 

ARTHUR  KLORFEIN  FUND 

AMOS,  WILLIAM,  University  of  Cambridge,  United  Kingdom 

HALS,  GARY,  Capital  University 

HUNTER,  JUDY,  Auburn  University 

LIGHT,  JEFFREY,  University  of  Colorado 

LIN,  PETER,  Johns  Hopkins  University 

OLIVERIRA,  ANA,  Universidade  Federal  Do  Rio  De  Janeiro,  Brazil 

WARD,  GARY,  University  of  California  at  San  Diego 

ALLEN  R.  MEMHARD  SCHOLARSHIP 
GROSOF,  DAVID,  Harvard  University 

JAMES  S.  MOUNTAIN  MEMORIAL  FUND,  INC.  SCHOLARSHIP 
HOLLINGSWORTH,  NANCY,  Oregon  State  University 

SOCIETY  OF  GENERAL  PHYSIOLOGISTS 

DONIACH,  TABITHA,  University  of  California  at  Santa  Cruz 

GRIMWADE,  BRIAN,  Yale  University 

LUTZ,  DOUGLAS,  University  of  Pennsylvania 


HONORS 


101 


FRANK  R.  LILLIE  FELLOWSHIP 
GUERRIER,  PIERRE  C.,  Station  Biologique  de  Roscoff,  France 

HERBERT  W.  RAND  FELLOWSHIP 

KAMIYA,  NOBURO,  National  Institute  for  Basic  Biology,  Japan 

JEAN  AND  KATSUMA  DAN  FELLOWSHIPS 

PRATT,  MELANIE  M.,  Harvard  University 

TANAKA,  YUICHIRO,  Sugashima  Marine  Biological  Station,  Japan 

MBL  AWARD  FOR  THE  MOST  OUTSTANDING  PAPERS  GIVEN  AT  THE  MBL 
GENERAL  SCIENTIFIC  MEETINGS  OF  AUGUST  1980 

"Coupling  between  Horizontal  Cells  in  the  Carp  Retina  Examined  by  Diffusion  of  Lucifer 
Yellow" 

By  AKIMICHI  KANEKO,  National  Institute  for  Physiological  Sciences,  Japan,  and  ANN  E. 
STUART,  University  of  North  Carolina 

"An  Optical  Determination  of  the  Resistance  in  Series  with  the  Axolemma  o/Loligo  pealei." 

By  BRIAN  M.  SALZBERG,  University  of  Pennsylvania,  FRANCISCO  BEZANILLA,  University 
of  California,  Los  Angeles,  and  H.  V.  DAVILA,  Universidad  Los  Andes,  Venezuela 


XIII.  INSTITUTIONS  REPRESENTED 


U.  S.  A. 


Alabama,  University  of,  Birmingham 

Albert  Einstein  College  of  Medicine 

Alfred  I.  duPont  Institute 

American  Cynamid  Company 

American  Museum  of  Natural  History 

Amherst  College 

Amray,  Inc. 

Anderson  &  Nichols 

Arizona,  University  of 

Arkansas,  University  of 

Armed  Forces  Radiobiology  Research 

Institution 
Atlanta  University 
Auburn  University 

Bates  College 

Baylor  College  of  Medicine 

Boston  Biomedical  Research  Institute 

Boston  University 

Boston  University  School  of  Medicine 

Bowdoin  College 

Bowman  Gray  School  of  Medicine 

Brandeis  University 

Bridgeport,  University  of 


Brown  University 

Bunting  Institute  of  Radcliffe  College 

California  State  College 
California,  University  of,  Berkeley 
California,  University  of,  Davis 
California,  University  of,  Irvine 
California,  University  of,  La  Jolla 
California,  University  of,  Los  Angeles 
California,  University  of,  Riverside 
California,  University  of,  San  Diego 
California,  University  of,  San  Francisco 
California,    University    of,    San    Francisco, 

Medical  School 

California,  University  of,  Santa  Barbara 
California,  University  of,  Santa  Cruz 
Capital  University 
Carl  Zeiss,  Inc. 

Carnegie  Institution  of  Washington 
Carnegie-Mellon  University 
Case  Western  Reserve  University 
Case  Western  Reserve  University  School  of 

Medicine 
Cathedral  High  School 


102 


MARINE  BIOLOGICAL  LABORATORY 


Chicago,  University  of 

Cincinnati,  University  of 

City  of  Hope  Medical  Center 

Claremont  Men's  College 

Clark  College 

Clark  University 

Clarkson  College  of  Technology 

Cleveland,  University  Hospitals  of 

Cold  Spring  Harbor  Laboratory 

Colorado  State  University 

Colorado,  University  of 

Colorado  Video 

Columbia  College 

Columbia  University 

Columbia  University,  College  of  Physicians 

and  Surgeons 
Connecticut  College 
Connecticut,  University  of 
Connecticut,  University  of,  Health  Center 
Conservation  Law  Foundation 
Cornell  Medical  College 
Cornell  University 
Council  on  Environmental  Quality 
Crimson  Camera  Technical  Sales,  Inc. 
C.  V.  Whitney  Laboratory 

DAGE-MTI 

Dartmouth  College 
Dartmouth  Medical  School 
Deep  Sea  Research 
Delaware,  University  of 
Denison  University 
Dillard  University 
Duke  University 

Duke  University  Medical  Center 
DuPont  Corporation 

Earlham  College 

East  Carolina  University,  Medical  School 

Eastman  Kodak  Company 

Eisenhower  College 

Emmanuel  College 

Emory  University 

Emory  University  School  of  Medicine 

Erskine  College 

Fairfield  University 

Florida  State  University 

Florida,  University  of,  College  of  Medicine 

General  Electric  Corporation 
George  Mason  University 
Georgia  Institute  of  Technology 
Georgia,  University  of 
Gonzaga  University 


Goucher  College 

Gray  Seal  Research  Station 

Hahnemann  Medical  College  and  Hospital 

Hamamatsu  Systems,  Inc. 

Hamilton  College 

Hanover  College 

Harvard  Medical  School 

Harvard  School  of  Public  Health 

Harvard  University 

Haverford  College 

Hawaii,  University  of 

Hawaii,  University  of,  Kewalo  Marine  Lab- 
oratory 

Hawaii,  University  of,  Pacific  Biomedical 
Research  Center 

Holy  Cross  College 

Howard  University 

Hunter  College 

Illinois  Institute  of  Technology 

Illinois,  University  of 

Illinois,  University  of,  College  of  Medicine 

Indiana  University 

Indiana  University  School  of  Medicine 

Institute  for  Cancer  Research,  The 

Institute  for  Scientific  Information 

Iowa  State  University 

Iowa,  University  of 

IWS  Seward  Marine  Station 

Jackson  State  University 

John  B.  Pierce  Foundation  Laboratory 

Johns  Hopkins  Hopsital 

Johns  Hopkins  University,  The 

Johns  Hopkins  University,  The,  School  of 

Hygiene  and  Public  Health 
Johns  Hopkins  University,  The,  School  of 

Medicine 
Johnson,  S.  C.  &  Son 

Kansas  State  University 
Kansas,  University  of 
Kresge  Eye  Institute 

Lab  Computer  Systems,  Inc. 

Laboratory  for  Comparative  Biochemistry 

Lafayette  College 

Lamont  Geological  Observatory 

Lehigh  University 

Leitz,  E.,  Inc. 

Life  Savers,  Inc. 

Louisiana  State  University 

Macalester  College 
Maine  Medical  Center 


INSTITUTIONS  REPRESENTED 


103 


Maine,  University  of 

Marine  Biomedical  Institute 

Marlboro  College 

Mary  Imogene  Bassett  Hospital 

Maryland,  University  of 

Maryland,  University  of,  School  of  Medicine 

Massachusetts  Eye  and  Ear  Infirmary 

Massachusetts  General  Hospital 

Massachusetts  Institute  of  Technology 

Massachusetts  State  Lobster  Hatchery 

Massachusetts,  University  of,  Amherst 

Medical  College  of  Ohio 

Mellon  Institute 

Merck  Institution  for  Therapeutic  Research 

Mercy  College 

MERIT  Fund,  Inc. 

Miami,  University  of 

Miami,  University  of,  School  of  Medicine 

Michigan  State  University 

Michigan,  University  of 

Milton  S.  Hershey  Medical  Center 

Minnesota,  University  of 

Minnesota,  University  of,  School  of  Medicine 

Miriam  Hospital 

Mississippi,  University  of,  Medical  School 

Missouri,  University  of 

Monell  Chemical  Senses  Center 

Montefiore  Hospital  and  Medical  Center 

Moravian  College 

Morehouse  College 

Mount  Holyoke  College 

Mount  Sinai  Hospital 

Nantucket  High  School 

National  Academy  of  Sciences 

National  Cancer  Institute 

National  Institute  of  Alcohol  Abuse  and  Al- 
coholism 

National  Institutes  of  Health 

National  Marine  Fisheries  Service/NOAA 

National  Resource  Defense  Council 

Nebraska,  University  of 

Neurological  Institute 

New  England  Aquarium 

New  England  Biolabs,  Inc. 

New  England  Medical  Center  Hospital 

New  Hampshire,  University  of 

New  Jersey  Medical  School 

New  York  Blood  Center 

New  York,  City  University  of,  Brooklyn  Col- 
lege 

New  York,  City  University  of,  City  College 

New  York,  City  University  of,  Herbert 
Lehman  College 

New  York,  City  University  of,  Hunter  Col- 
lege 


New  York  Institute  for  Basic  Research  in 

Mental  Health 
New  York  Medical  College 
New  York  State  College  of  Veterinary  Med- 
icine 

New  York,  State  University  of,  Albany 
New  York,  State  University  of,  Binghamton 
New  York,  State  University  of,  Buffalo 
New  York,  State  University  of,  Downstate 

Medical  Center 

New  York,  State  University  of,  New  Paltz 
New  York,  State  University  of,  Stony  Brook 
New  York,  State  University  of,  Syracuse 
New  York  University  Medical  Center 
Nichols  College 
Nikon,  Inc. 

North  Carolina  State  University 
North  Carolina,  University  of,  Chapel  Hill 
North  Carolina,  University  of,  Charlotte 
Northeastern  Ohio  Universities  College  of 

Medicine 

Northeastern  University 
Northwestern  University 
Northwestern  University  Medical  School 
Notre  Dame,  University  of 

Oak  Ridge  National  Laboratories 

Oberlin  College 

Ohio  University 

Ohio  University  College  of  Medicine 

Ohio  Wesleyan  University 

Oklahoma  State  University 

Olympus  Corporation  of  America 

Opti-Quip,  Inc. 

Oregon  State  University 

Oregon,  University  of 

Pace  University 

Pennsylvania,  Medical  College  of 

Pennsylvania  State  University 

Pennsylvania,  University  of 

Pennsylvania,  University  of,  School  of  Dental 
Medicine 

Pennsylvania,  University  of,  School  of  Med- 
icine 

Pittsburgh,  University  of 

Pittsburgh,  University  of,  School  of  Medicine 

Polaroid  Corporation 

Polaron  Instruments  Inc. 

Pomona  College 

Princeton  University 

Public  Health  Research  Institute 

Purdue  University 

Rainbow  Babies  and  Children's  Hospital 
Rhode  Island  College 


104 


MARINE  BIOLOGICAL  LABORATORY 


Rhode  Island,  University  of 

Riley  Hospital  for  Children 

Rochester,  University  of 

Rochester,  University  of,  School  of  Medicine 

Rockefeller  Foundation 

Rockefeller  University,  The 

Roosevelt  University 

Rush  Medical  College 

Rush-Presbyterian-St.  Luke's  Medical  Cen- 
ter 

Rush  University 

Rutgers — The  State  University  of  New  Jer- 
sey 

Rutgers  University  Medical  School 

St.  Elizabeth's  Hospital 

St.  Jude's  Children's  Research  Hospital 

St.  Louis  University  School  of  Medicine 

St.  Peter's  College 

St.  Vincent  Charity  Hospital 

San  Diego  State  University 

Santa  Fe,  College  of 

Savannah  River  Ecology  Program 

Scripps  Institution  of  Oceanography 

Sea  World's  Shark  Institute 

Seiler  Instruments 

Sidney  Farber  Cancer  Institute 

Smithsonian  Institution 

Southern  Connecticut  State  College 

Southern  Florida,  University  of 

Southern  Florida,  University  of,  Medical 
Center 

Stanford  University 

Stanford  University,  Hopkins  Marine  Sta- 
tion 

Stanford  University,  School  of  Medicine 

Stetson  University 

Swarthmore  College 

Sweet  Briar  College 

Syracuse  University 

Temple  University  Medical  School 

Tennessee,  University  of 

Texas  Medical  Center 

Texas  Southern  University 

Texas,  University  of 

Texas,  University  of,  Health  Science  Center 

Texas,  University  of,  Medical  Branch 

Thomas  Jefferson  University 

Thomas  Jefferson  University  Medical  School 

Tougaloo  College 


Towson  State  University 
Tufts  University 

Tufts  University  Medical  School 
Tufts  University  School  of  Veterinary  Med- 
icine 
Tulane  University 

Union  College 

United  States  Coast  Guard 

United  States  Department  of  Agriculture 

United    States     Environmental    Protection 

Agency 
United  States  Forest  Service 

Vanderbilt  University 

Vanderbilt  University  School  of  Medicine 

Vassar  College 

Venus  Scientific,  Inc. 

Vermont,  University  of 

Veterans  Administration  Medical  Center, 
Boston 

Vickers  Instruments 

Villanova,  University  of 

Virginia  Polytechnic  Institute  and  State  Uni- 
versity 

Virginia,  University  of 

Viterbo  College 

Washington  University 
Washington,  University  of 
Washington  University,  School  of  Medicine 
Wayne  State  University 
Wayne  State  University,  School  of  Medicine 
Wesleyan  University 
West  Chester  State  College 
West  Virginia  University 
Williams  College 
Wisconsin,  University  of 
Wistar  Institute 
Wofford  College 

Woods  Hole  Oceanographic  Institution 
Worcester  Foundation  for  Experimental  Bi- 
ology 

Worcester  State  College 
World  Wildlife  Fund 

Xavier  University 

Yale  University 

Yale  University,  School  of  Medicine 

Yeshiva  College 


FOREIGN  INSTITUTIONS 


Academia  Sinica,  People's  Republic  of  China 
Alberta,  University  of,  Canada 


Athens,  University  of,  Greece 
Australian  National  University,  Australia 


INSTITUTIONS  REPRESENTED 


105 


Basel,  University  of,  Switzerland 

Bedford  Institute  of  Oceanography,  Canada 

Berlin,  Free  University  of,  West  Germany 

Bermuda  Biological  Station,  England,  UK 

Brazil,  University  of,  Brazil 

Calgary,  University  of,  Canada 

Cambridge,  University  of,  England,  UK 

Cell  Biology  Laboratory,  Italy 

Centro  de  Ciencias  del  Mar  y  Limnologia, 

Mexico 
Centro  de  Investigacion  y  de  Estudios  Avan- 

zados,  Mexico 

Claude  Bernard,  University  of,  France 
Conservation  de  Paris,  France 
Copenhagen,  University  of,  Denmark 
Dalhousie  University,  Canada 
East  Anglia,  University  of,  England,  UK 
Faculte  de  Medecine,  France 
F.I. D.I. A.  Research  Laboratories,  Italy 
Hebrew  University  of  Jerusalem,  Israel 
Hebrew  University  Medical  School,  Israel 
Institut  fur   Mikrobiologie   Zoologie,   West 

Germany 

Institute  Ferreyra,  Argentina 
Institute  of  Molecular  Biology,  Austria 
Institution   for  Molecularbiologie,  Switzer- 
land 

Institute  de  Biofisica,  Brazil 
Institute  de  Investigacion  Medica,  Argentina 
Institute  Politecnico  Nacional,  Mexico 
Institute     Venezolano     de     Investigaciones 

Cientificas,  Venezuela 

International  Institute  of  Genetics  and  Bio- 
physics, Italy 
International   Laboratory   for   Research  on 

Animal  Disease,  Kenya 
Korea  Advanced   Institute  of  Science  and 

Technology,  Kenya 

Laboratory    de     Neurobiologie    Cellularie, 
France 


Laval  University,  Canada 
Liverpool  School  of  Tropical  Medicine,  Eng- 
land, UK 

Max-Planck  Institute,  West  Germany 
McGill  University,  Canada 
McMaster  Medical  Center,  Canada 
Medical  Research  Council,  UK 
Melbourne,  University  of,  Australia 
Montreal  General  Hospital 
Mount  Allison  University,  Canada 
National  Institute  for  Basic  Biology,  Japan 
National  Institute  for  Medical  Research,  UK 
North  Wales,  University  College  of,  Great 

Britain,  UK 

Ottawa,  University  of,  Canada 
Oxford,  University  of,  England,  UK 
Paris,  University  of,  France 
Puerto  Rico,  University  of 
Scarborough  College,  Canada 
Sri  Lanka,  University  of,  Sri  Lanka 
State  Fisheries  Laboratory,  Mexico 
Station  Biologique,  France 
Stazione  Zoologici  di  Napoli,  Italy 
Stockholm,  University  of,  Sweden 
Sugashima  Marine  Biological  Station,  Japan 
Technical  Medical  School,  Israel 
Tel  Aviv  University,  Israel 
Tokyo,  University  of,  Japan 
Torino,  University  of,  Italy 
Toronto,  University  of,  Canada 
Universidad  Federal  de  Rio  de  Janeiro,  Bra- 
zil 
Universidad  Nacional  Autonoma  de  Mexico, 

Mexico 

Universidad  Peruana  Cayetano  Heredia,  Peru 
University  School  of  Medicine,  Japan 
Utrecht,  University  of,  Netherlands 
Wellcome  Research  Laboratories,  UK 


XIV.  LABORATORY  SUPPORT  STAFF 

Including  Persons  Who  Joined  or  Left  the  Staff  During  1981 


Controller's  Office 

CASEY,  EDWARD  G.,  Controller 
CAMPBELL,  RUTH  B. 
DAVIS,  DORIS  C. 


ELLIS,  NANCY  L. 
HOBBS,  ROGER  W.,  JR. 
WALSH,  MICHAEL  P. 


Director's  Office 

GROSS,  PAUL  R.,  President  and  Director 
THIMAS,  LISA  M. 


VALANTI,  IRIS 


106 


MARINE  BIOLOGICAL  LABORATORY 


General  Manager's  Office 

SMITH,  HOMER  P.,  General  Manager 
BAKER,  KAREN  H. 
BUTZ,  FLORENCE  S. 
GEGGATT,  AGNES  L. 


JOHNSON,  FRANCES  N. 
McGoLDRiCK,  MICHELLE 

POMERLEAU,  LlNDA  M. 


Associate  Director's  Office 

PALMER,  DOUGLAS  W.,  Associate  Director       ZIEMER,  CAROL  A. 

Education  Office 

MASER,  MORTON  D,  Assistant  Director  for  Educational  and  Research  Services 

ALLEN,  GENEVIEVE 

LEIGHTON,  JANE  L.,  Admissions  Officer  ST.  HILAIRE,  LYNDA  D. 


Grants  Office 

HOWARD,  JOAN  E.,  Grants  Officer 


ROLAND,  RENEE 


Public  Relations  Office 

HASKELL,  BARBARA,  Public  Relations  Officer 
CAMPBELL,  LEE  ANNE 


Biological  Bulletin 

METZ,  CHARLES  B.,  Editor 
LANG,  HELEN  E. 


MOUNTFORD,  REBECCA 
SCHWARTZ,  SUSAN 


Buildings  and  Grounds 

GUNNING,  A.  ROBERT,  Superintendent 

ANDERSON,  LEWIS  B. 

BERRIOS,  JOSE  R. 

BOURGOIN,  LEE  E. 

BRODERICK,  MADELINE 

CAFARELLI,  PETER  A. 

CARINI,  ROBERT  J. 

COSTA,  ROBERT  A. 

DAVIS,  MARK  A. 

DUTRA,  STEVEN  M. 

ENOS,  GLENN  R. 

FISH,  STEPHEN 

FUGLISTER,  CHARLES  D. 

GEGGATT,  RICHARD  E.,  JR. 

GIBBONS,  ROBERTO  G. 

GONSALVES,  WALTER  W.,  JR. 

KLEINDINST,  THOMAS  N. 

KUIL,  ELISABETH 


LEHY,  DONALD  B. 
LEWIS,  RALPH  H. 
LOCHHEAD,  WILLIAM  M. 
LOVERING,  RICHARD  A. 
LUNN,  ALAN  G. 
MACLEOD,  JOHN  B. 
MILLS,  STEPHEN  A. 
MOORE,  MORGAN 
PETERSON,  PHILIP 
ST.  JEAN,  SIMONE 
SMART,  MERILYN  A. 
TAVARES,  GENE 
THRASHER,  FREDERICK 
VARAO,  JOHN 
WARD,  FREDERICK 
WEEKS,  GORDON  W. 
WHITTAKER,  WILLIAM 


Gray  Museum 

TIFFNEY,  WESLEY  N.,  Curator 
BORETOS,  C.  DIANE 
BUSH,  LOUISE 


MILLS,  SUSAN  W. 
MONTIERO,  EVA  S. 
MOUL,  EDWIN  T. 


LABORATORY  SUPPORT  STAFF 


107 


Library 

FESSENDEN,  JANE,  Librarian 
ASHMORE,  JUDITH  A. 
CLARK,  SARAH 
COOMBS,  ROXANNE 
DEVEER,  JOSEPH  M. 
FITZGERALD,  DAVID  J. 
GIBBONS,  ROBERTO  G. 
GRICE,  JOAN  H. 
HANLEY,  JANICE  S. 


HOUGH,  NANCY  L. 
IRVING,  LYNNE  A. 
JOSEPH,  E.  LENORA 
MARGOLIN,  JILL 
MOUNTFORD,  REBECCA  J. 
NORTON,  CATHERINE 
SWAIN,  LAUREL 


Marine  Resources 

VALOIS,  JOHN  J.,  Manager 
ENOS,  EDWARD  G.,  JR. 
ENOS,  JOYCE 
HEBDEN,  ROBERT  M. 
LAWDAY,  LEWIS  M. 
MURPHY,  CHARLES 

Research  Services 

MASER,  MORTON  D,  Assistant  Director  for 

Educational  and  Research  Services 
BALDIC,  DAVID 
BARNES,  FRANKLIN  D. 
BARNES,  JOHN  S. 
EVANS,  WILLIAM 
COLDER,  LINDA  M. 
GOLDER,  ROBERT  J. 
KERR,  Louis 

Summer  Support  Staff 

ALBERS,  CHRISTINA 
ANDERSON,  JANICE  A. 
ASCI,  MARGUERITE  M. 
ASHMORE,  MICHAEL  W. 
BLACK,  ROBERT  W. 
BLAIR,  RICHARD  H. 
BLOODWORTH,  BAKER 
BODZNICK,  MARLA 
BRINKMAN,  PAULA  M. 
BROADSTOCK,  PENELOPE 
BURNETT,  LYNN 
CHILD,  MALCOLM 
COOMBS,  GILLIAN 
CRUISE,  MEGHAN  F. 
EMMONS,  BARRY 
FENNELLY,  GLENN  J. 
FITE,  DAVID 
FITZGERALD,  MARY  E. 
GIFFORD,  HEIDI 
GRASS,  ANTHONY 
GROSSMAN,  ROBERT 
GUNTER,  SUSAN 
HANSON,  ANTHONY 
HARDING,  RICHARD 


SMITH,  A.  DICKSON 
TASSINARI,  EUGENE 
TRAPASSO,  BRUNO 
VARAO,  JOHN 


MAKREDES,  NICHOLAS 
MARTIN,  LOWELL  V. 
NICHOLS,  FRANCIS  H.,  JR. 
SILVA,  MARK 
SYLVIA,  FRANK  E. 
VARAO,  JOHN 


IRISH,  BRADFORD  G. 
JOHNSON,  JEFFREY 
KELLY,  KEVIN 
KULLBERG,  PAULA  G. 
LEE,  JAMES  M. 
LEONARD,  LESLIE  ANNE 
LUNN,  JEFFREY 
MAXWELL,  BRETT 
MLODZINSKA,  KATHERINE  M. 
MONIZ,  KAREN 
PARKER,  TIMOTHY 
PIERCE,  RICHARD 
ROONEY,  CARRIE 
ROONEY,  COLLEEN 
ROSCOE,  JOAN  E. 
SMITH,  NATHAN  B. 
SOUZA,  JACQUELINE  L. 
STONE,  AMY 
TRIPP,  TERRY 
VALOIS,  FRANCIS 
WALKER,  ALLEN  R. 
WHITTAKER,  WILLIAM  A. 

WUETHRICH,  LISA 

WYTTENBACH,  ROBERT  A. 


Reference:  Biol.  Bull.  163:  108   130.  (August,  1982) 


FISH  GILL  IONIC  TRANSPORT:  METHODS  AND  MODELS 

DAVID  H.  EVANS',  J.  B.  CLAIBORNE2t,  LINDA  FARMER2,  CHARLES  MALLERY2,  AND 

EDWARD  J.  KRASNY,  JR.3 

Department  of  Zoology,  University  of  Florida,  Gainesville,  FL  32611;  2 Department  of  Biology, 
University  of  Miami,  Coral  Gables,  FL  33124;  and  ^Department  of  Physiology  and  Biophysics, 

University  of  Alabama,  Birmingham,  AL  35294 

INTRODUCTION 

Because  fishes,  like  all  aquatic  vertebrates  except  the  marine  hagfishes,  maintain 
the  Na+  and  Cl~  content  of  their  body  fluids  distinctly  different  from  either  their 
freshwater  or  marine  environment,  they  face  a  constant  net  movement  of  salts  and 
water*  across  their  permeable  membrane  (predominantly  the  branchial  epithe- 
lium). Thus,  freshwater  fishes  (which  are  hyperosmotic  to  the  medium)  presumably 
face  a  net  loss  of  NaCl  and  net  influx  of  water.  The  reverse  is  presumably  true  for 
the  hypo-osmotic  marine  fishes,  i.e.  they  face  a  net  gain  of  NaCl  and  loss  of  water. 
The  general  mechanisms  which  fishes  utilize  to  balance  these  net  salt  and  water 
movements  were  first  outlined  by  Homer  Smith  (1930)  and  have  been  more  recently 
reviewed  rather  extensively  (Potts  and  Parry,  1964;  Maetz,  1974;  Kirschner,  1977, 
1979;  Evans,  1979,  1980a).  In  the  past  few  years  it  has  become  increasingly  obvious 
that  rather  complex,  but  quite  intriguing,  mechanisms  of  ion  transport  are  resident 
in  the  epithelium  of  at  least  the  teleost  fish  gill**  (Maetz  and  Bornancin,  1975; 
Maetz  el  al.,  1976;  Kirschner,  1977,  1979,  1980;  Potts,  1977;  Evans,  1979,  1980b, 
1982a). 

It  is  not  the  aim  of  the  present  review  to  carefully  re-examine  the  data  which 
have  been  discussed  in  these  reviews.  Instead,  we  propose  to  examine  some  recent 
techniques  which  have  been  employed  to  attempt  to  more  carefully  delineate  the 
mechanisms  of  fish  branchial  ionic  transport.  In  the  process  we  intend  to  describe 
what,  and  what  not,  these  techniques  can  tell  us  about  this  system  as  well  as  what 
we  think  we  know  about  the  various  ionic  transport  mechanisms,  and  where  we 
think  we  should  be  heading  in  the  future. 

Since  many  of  the  techniques  have  been  developed  in  the  past  20  years  to  avoid 
some  of  the  problems  of  in  vivo  studies,  it  is  appropriate  to  begin  with  a  description 
of  whole  animal  kinetic  and  electrochemical  studies. 

Received  22  February  1982;  accepted  21  May  1982. 

t  Present  address:  Abteilung  Physiology,  Max  Planck  Institut  fur  Experimentelle  Medizin,  Gottin- 
gen,  Federal  Republic  of  Germany. 

Abbreviations:  IPHP,  isolated,  perfused  head  preparation;  TAP,  triaminopyrimidine;  TEP, 
transepithelial  potential. 

*  Since  the  eiasmobranch  fishes  (sharks,  skates,  etc.}  maintain  isotonicity  to  sea  water  via  the 
retention  of  urea,  they  are  an  exception  to  this  statement  about  net  movements  of  water.  However,  it 
is  important  to  note  that  their  body  fluids  contain  significantly  less  NaCl  than  sea  water,  so  they  still 
face  net  influxes  of  these  ions  in  sea  water  (see  Evans,  1979,  for  a  more  complete  examination  of 
eiasmobranch  osmoregulation). 

**  Henceforth  we  will  he  dealing  with  teleost  fish,  with  only  slight  reference  to  the  elasmobranchs, 
until  the  last  section  of  this  review. 


108 


FISH  GILL  IONIC  TRANSPORT  109 

THE  INTACT  ANIMAL 

Investigations  of  possible  mechanisms  of  gill  transport  actually  started  when 
Krogh  (1939)  demonstrated  that  the  head  end  of  goldfish  (Carassius  auratus)  was 
able  to  extract  Na+  and  Cl~  from  solutions  independently  of  each  other.  He  pro- 
posed that,  since  the  uptake  mechanisms  were  parallel  but  uncoupled,  there  were 
probably  ionic  exchange  systems  involved  to  maintain  some  semblance  of  electro- 
neutrality.  He  suggested  that  the  Na+  uptake  might  be  coupled  to  NH4+  extrusion 
and  that  Cl~  uptake  might  be  coupled  to  HCO3  efflux.  In  an  early  study  utilizing 
radioisotopes  Maetz  and  Garcia  Romeu  (1964)  supported  Krogh's  proposition  by 
demonstrating  that  Na+  and  Cl~  uptake  could  be  stimulated,  independently  of  each 
other,  by  injecting,  respectively,  NH4+  or  HCO3~  into  the  blood  of  the  goldfish. 
Addition  of  the  same  substances  to  the  external  medium  inhibited  Na+  and  Cl" 
uptake,  respectively.  Since  the  uptakes  could  be  measured  with  22Na  and  36C1,  this 
study  represented  a  significant  step  forward  from  earlier  studies  which  relied  on 
chemical  analysis  to  monitor  net  fluxes.  Nevertheless  it  also  demonstrates  at  least 
two  pitfalls  of  the  majority  of  whole-animal  studies,  even  to  the  present  time.  Na+ 
and  Cl  are  ions  and  their  movements  are  therefore  affected  by  electrical  potentials 
as  well  as  chemical  gradients.  Therefore  it  is  possible  that  an  experimental  ma- 
nipulation (in  this  case  ionic  substitution)  which  results  in  an  alteration  of  the 
movement  of  an  ion  produces  this  change,  not  through  a  direct  effect  on  some  sort 
of  ionic  exchange  system,  but  by  altering  the  electrical  gradient  (transepithelial 
potential,  TEP)  across  the  epithelium  in  question.  In  other  words,  in  the  Maetz 
and  Garcia  Romeu  (1964)  experiments,  it  is  possible  (for  example)  that  injection 
of  (NH4)2SO4  altered  some  TEP  across  the  fish,  made  the  blood  more  electroneg- 
ative (relative  to  the  fresh  water)  and  thereby  stimulated  a  passive  uptake  of  Na+. 
Corresponding  arguments  could  be  applied  to  the  effects  of  KHCO3  injections  on 
Cl"  uptake  and  the  effects  of  external  additions  of  either  substance  on  Na+  and 
Cl"  uptake.  Thus,  firm  statements  about  chemical  vs  electrical  couplings  simply 
cannot  be  made  without  concomitant  measurements  of  the  TEP. 

The  other  pitfall  of  whole-animal  studies  (as  demonstrated  by  the  early  work 
of  Maetz  and  Garcia  Romeu,  1964)  is  that  specific  alteration  of  the  composition 
of  the  blood  of  intact  animals  is  nearly  impossible.  For  example  the  injection  of 
(NH4)2SO4  into  the  blood  could  have  lowered  the  pH  of  the  blood  and  thereby 
stimulated  a  Na+/H+  rather  than  Na+/NH4+  exchange  as  proposed  by  the  authors. 
Kerstetter  el  al.  (1970)  noted  this  potential  and  found  that  stimulation  of  Na+ 
uptake  (produced  by  increasing  the  external  Na+  concentrations)  was  correlated 
with  a  stimulation  of  acid  efflux,  rather  than  ammonia  efflux.  In  addition,  in  this 
study,  TEPs  were  monitored  and  shown  to  be  insufficient  to  account  for  the  increase 
in  flux  of  either  Na+  or  acid.  Interestingly,  the  study  by  Kerstetter  et  al.  (1970) 
demonstrates  another  drawback  of  whole-animal  studies.  They  injected  the  carbonic 
anhydrase  inhibitor  acetazolamide  into  trout  and  found  that  both  Na+  uptake  and 
acid  efflux  was  inhibited.  While  these  data  could  support  the  proposition  that  Na+/ 
H+  exhange  is  present  and  limited  by  the  production  of  protons  by  the  hydration 
of  CO2  in  the  branchial  cells,  the  fall  in  both  fluxes  could  have  been  secondary  to 
cardiovascular  effects  of  the  injected  drug.  That  ammonia  efflux  did  not  change 
significantly  during  the  same  treatment  argues  against  general  cardiovascular  ef- 
fects, but  it  is  possible  that  ammonia  efflux  is  via  a  pathway  which  is  relatively 
unaffected  by  cardiovascular  changes.  The  unfortunate  fact  is  that  whole-animal 
studies  do  not  allow  the  separation  of  cardiovascular  from  epithelial  effects. 


110  DAVID  H.  EVANS  ET  AL. 

Thus,  attempts  to  manipulate  blood  ionic  concentrations,  injection  of  potentially 
cardiovascular-active  drugs,  and  lack  of  monitoring  of  the  TEP  present  pitfalls 
which  could  bring  into  question  the  conclusions  of  many  whole-animal  studies. 
Some  of  these  problems  can  be  avoided  by  using  externally  applied  drugs  or  by 
monitoring  the  efflux  of  an  ionic  species  when  ionic  substitutions  are  made  in  the 
external  medium.  If  one  assumes  that  external  addition  of  drugs  or  ionic  substi- 
tutions do  not  have  cardiovascular  effects,  and  one  monitors  the  TEP,  many  of  the 
problems  of  earlier  studies  can  be  avoided.  For  example,  we  have  recently  found 
(Evans,  1977,  1982b;  Evans  et  al.,  1979)  that  various  species  of  marine  teleost  and 
elasmobranch  fishes  excrete  ammonia  and  H+,  and  that  approximately  50%  of  the 
ammonia  efflux  and  100%  of  the  H+  efflux  is  dependent  on  external  Na+.  Mea- 
surements of  the  TEP  indicate  that  the  coupling  is  not  electrical.  It  is  interesting 
to  note  that  the  excretion  of  ammonia  from  intact  freshwater  fishes  is  relatively 
unaffected  by  the  removal  of  external  Na+  (deVooys,  1968;  Kerstetter  et  al.,  1970; 
Maetz,  1973).  Unfortunately,  only  Kerstetter  et  al.  (1970)  monitored  the  TEP, 
and  they  found  that  TEP  changes  were  insufficient  to  account  for  any  flux  changes, 
or  lack  thereof. 

The  data  from  intact  fish  on  C1~/HCO3~  exchange  is  more  sparse,  but  less 
equivocal.  Dejours  (1969)  found  that  when  the  external  medium  of  a  goldfish  was 
changed  from  NaCl  to  Na2SO4,  CO2  excretion  fell  to  zero  and  was  restimulated 
when  the  fish  was  again  placed  into  NaCl  solutions.  In  addition,  DeRenzis  and 
Maetz  (1973)  demonstrated  a  good  correlation  between  the  uptake  of  Cl~  and  the 
net  excretion  of  base  by  the  goldfish,  and  DeRenzis  (1975)  found  that  addition  of 
thiocyanate  to  the  external  bath  inhibited  Cl~  uptake  and  base  excretion.  Unfor- 
tunately, only  in  the  latter  study  were  TEPs  measured,  but  they  could  not  account 
for  the  effect. 

In  most  cases  whole-animal  studies  treat  the  branchial  transporting  cells  as 
"black  boxes"  and  cannot  separate  events  taking  place  on  the  basolateral  vs  apical 
surfaces  of  the  cells.  For  example,  the  fact  that  removal  of  external  Na+  inhibits 
50%  or  less  of  the  ammonia  efflux  from  marine  fish  supports  the  proposition  (Evans, 
1977)  that  Na+/NH4+  exchange  is  taking  place,  but  it  does  not,  in  itself,  indicate 
the  site  of  this  ionic  exchange  system.  Maetz  and  Garcia  Romeu  (1964)  found  that 
the  carbonic  anhydrase  inhibitor  acetazolamide  inhibited  both  Na+  and  Cl~  uptake 
when  injected  into  the  blood  of  the  goldfish.  They  proposed  that  both  Na+  and  Cl~ 
uptake  must  therefore  be  limited  by  the  production  of  H+  (for  the  protonation  of 
NH3  which  had  been  produced  in  the  branchial  cells)  and  HCO3~.  Since  Na+  and 
Cl~  are  taken  up  independently  from  extremely  low  salinities  (in  micromolar  ranges 
in  some  cases),  and  probably  exchanged  for  these  intracellular  electrolytes  (see 
above),  it  seems  most  appropriate  to  propose  that  the  ionic  exchange  systems  are 
on  the  apical  border  of  the  transporting  cell  (Maetz  and  Garcia  Romeu,  1964; 
Kirschner,  1977,  1979).  This  model  (Fig.  1)  is  supported  by  the  finding  that  ami- 
loride  (which  is  known  to  inhibit  uptake  of  Na+  in  a  wide  variety  of  tissues  (Cuthbert 
et  al.,  1979))  also  inhibits  both  ammonia  and  acid  efflux  from  fish  (Kirschner  et 
al.,  1973). 

The  potential  problems  of  whole-animal  studies  are  especially  evident  when  one 
examines  the  history  of  the  study  of  Na+  and  Cl~  extrusion  mechanisms  in  marine 
species.  A  more  extensive  discussion  of  this  subject  was  presented  elsewhere  (Evans, 
1979).  In  the  late  1960's  it  was  found  that  the  marine  teleost  branchial  epithelium 
contained  significant  quantities  of  the  enzyme  (Na+-K+  activated  ATPase)  which 
mediated  Na+/K+  exchange  in  a  variety  of  tissues,  and  that  these  enzyme  activity 
levels  were  lower  in  freshwater  species  and  after  freshwater  adaptation  of  euryhaline 


FISH  GILL  IONIC  TRANSPORT 


111 


BLOOD 


co2- 


NH4 
or 
NH3- 


C.A, 


H 


FRESH 
WATER 


FIGURE  1.  Tentative  model  for  mechanisms  of  Na+  and  Cl  uptake  by  the  branchial  epithelium 
of  freshwater  fishes.  Redrawn  from  Maetz  and  Garcia  Romeu  (1964).  See  text  for  details,  supporting 
evidence,  and  additions. 


species  (Epstein  et  al.,  1967).  Soon  thereafter  it  was  discovered  that  the  efflux  of 
radiosodium  from  the  eel,  Anguilla  anguilla,  was  sensitive  to  the  external  (sea- 
water)  concentration  of  K+  (Maetz,  1969).  Thus,  it  appeared  that  the  seawater  fish 
gill,  like  so  many  other  tissues,  extruded  unwanted  Na+  in  exchange  for  seawater 
K+,  utilizing  the  enzyme  Na+-K+  activated  ATPase  (Maetz,  1971). 

This  model  was  strengthened  by  the  finding  that  another  species  of  marine 
teleost  (the  fat  sleeper,  Dormitator  maculatus}  also  possessed  a  K+-sensive  Na+ 
efflux,  with  a  K+  sensitivity  (delineated  by  the  Km  =  2  mM  K+)  identical  to  that 
of  the  Na+-K+  activated  ATPase  extracted  from  the  gill  tissue  (Evans  et  al.,  1973). 
In  addition,  the  time  course  of  activation  of  the  enzyme  was  identical  to  the  time 
course  of  activation  of  the  K+  sensitive  Na+  efflux  when  this  species  was  transferred 
from  fresh  water  to  sea  water  (Evans  and  Mallery,  1975).  However  pleasant  this 
model  for  Na+  extrusion  was,  it  rapidly  became  apparent  that  the  system  was  much 
more  complex.  These  initial  studies  neglected  to  measure  the  TEP  during  these 
ionic  substitutions.  Indeed,  earlier  studies  (House,  1963;  Evans,  1969)  had  dem- 
onstrated that  the  TEP  across  two  species  of  marine  teleosts  was  nearly  identical 
to  the  equilibrium  potential  for  Na+,  i.e.,  Na+  was  possibly  in  passive  equilibrium 
across  the  fish  gill  because  the  prevailing  chemical  gradient  favoring  net  diffusional 
gain  was  balanced  by  a  blood-positive  (to  sea  water)  electrical  potential  of  sufficient 
magnitude  to  balance  the  chemical  gradient.  Thus,  these  data  indicated  that  the 
net  salt  gain  in  sea  water  was  not  NaCl,  but  only  Cl"  because  Na+  was  in  elec- 
trochemical equilibrium.  This  idea  has  been  at  least  partially  substantiated  by  more 
recent  whole-animal  TEP  determinations;  however,  it  has  been  found  that  some 


112 


DAVID  H.  EVANS  ET  AL. 


BLOOD 


Cl 


SEA 
WATER 


Cl  . 


No 


-  No 


FIGURE  2.    Current  model  for  the  mechanisms  of  Na+  and  Cl    extrusion  by  the  branchial  epithelium 
of  marine  fishes.  Redrawn  from  Silva  et  al.  (1977).  See  text  for  details  and  supporting  evidence. 


species  apparently  maintain  TEPs  distinctively  below  the  equilibrium  potential  for 
Na+  (see  reviews  by  Kirschner,  1979,  1980,  and  Evans,  1980b).  If  Na+  is  in  elec- 
trochemical equilibrium  then  the  ionic  substitution  experiments  which  indicated 
that  Na+/K+  exchange  may  be  taking  place  may  also  possibly  be  explained  by 
TEP  changes.  This  has  proved  to  be  the  case  in  some  species  (Potts  and  Eddy, 
1973;  Kirschner  et  al.,  1974)  but  not  in  others  (Evans,  1975;  Maetz  and  Pic,  1975; 
Evans  and  Cooper,  1976).  In  addition,  Na+/Na+  exchange  diffusion  which  was 
first  described  by  Motais  et  al.  (1966)  has  now  been  shown  to  be  a  TEP  effect  in 
some  species  (Potts  and  Eddy,  1973;  Kirschner  et  al.,  1974)  but  not  others  (Evans, 
1975;  Maetz  and  Pic,  1975;  Evans  and  Cooper,  1976). 

Thus,  whole-animal  studies  on  the  mechanisms  for  salt  extrusion  in  sea  water 
have  left  us  with  the  rather  unsatisfying  conclusion  that  some  animals  may  be 
extruding  net  amounts  of  Na+  and  Cl  and  others  may  only  need  to  extrude  Cl~ 
(the  TEP  of  all  marine  teleosts  examined  to  date  is  distinctly  different  from  the 
equilibrium  potential  for  Cl~;  Evans,  1980b).  Studies  of  the  mechanisms  of  Cl~ 
extrusion  by  whole  animals  have  indicated  that  it  is  sensitive  to  the  external  K+ 
concentration  (Epstein  et  al.,  1973)  and  to  the  external  HCO3  but  not  OH~ 
concentration  (Kormanik  and  Evans,  1979),  and  inhibited  by  injection  of  thiocy- 
anate  (Epstein  et  al.,  1973).  In  addition,  both  Na+  and  Cl~  efflux  are  inhibited  by 
injection  of  the  Na+-K+  activated  ATPase  inhibitor  ouabain  into  the  blood  of  the 
eel,  Anguilla  rostrata  (Silva  et  al.,  1977).  Since  it  had  been  shown  that  Na+-K+ 
activated  ATPase  is  actually  located  on  the  basolateral  plasma  membranes  (Kar- 
naky  et  al.,  1976),  these  authors  proposed  that,  like  many  other  tissues  (Frizell  et 
al.,  1979),  the  marine  teleost  gill  epithelium  secretes  Cl  via  a  basolateral  co- 


FISH  GILL  IONIC  TRANSPORT  1  1  3 

transport  of  Na+  and  Cl~  (energized  by  the  movement  of  Na+  down  its  electro- 
chemical gradient,  which  is  maintained  by  Na+-K+  activated  ATPase),  followed 
by  movement  of  Cl~  down  its  electrochemical  gradient  from  the  cell  to  the  sea 
water.  Na+  is  maintained  in  electrochemical  equilibrium.  This  model  (Fig.  2)  cer- 
tainly goes  far  to  explain  most  of  the  present  data  from  intact  animals,  but,  of 
course,  does  not  explain  Na+  extrusion  by  fish  which  have  been  shown  to  maintain 
Na+  out  of  electrochemical  equilibrium  (see  review  by  Evans,  1980b).  The  sensi- 
tivity to  external  HCO3  (Kormanik  and  Evans,  1979)  is  also  not  explained  by  this 
model. 

Unfortunately,  the  use  of  intact  animals  precludes  the  most  obvious  experiments 
to  test  this  interesting  model.  Ouabain  is  a  potent  cardiovascular  agent  and,  indeed, 
Silva  et  al.  (1977)  did  find  that  even  the  efflux  of  tritiated  water  from  A.  rostrata 
declined  by  some  40%  after  the  injection  of  sufficient  ouabain  to  produce  a  plasma 
concentration  of  2.5  X  10~6  M.  This  presumably  represented  some  sort  of  alteration 
in  blood  flow  through  the  branchial  vasculature,  which  in  theory  could  have  had 
a  more  pronounced  effect  of  the  efflux  of  both  Na+  and  Cl~  than  tritiated  water. 
Thus,  the  fact  that  ouabain  treatment  inhibited  Na+  and  Cl~  efflux  by  90%  does 
not  necessarily  prove  a  direct  effect  on  a  basolateral  uptake,  dependent  upon  a 
functioning  Na+/K+  exchange.  In  addition,  one  cannot  specifically  remove  blood 
Na+  to  examine  the  effect  on  Cl~  efflux  (according  to  the  Silva  model,  it  would 
decline  significantly). 

In  the  past  few  years,  whole-animal  studies  have  been  utilized  to  demonstrate 
that  the  Na+/H+  or  NH4+  exchange  which  characterizes  freshwater  fish  ion  reg- 
ulation is  also  present  in  marine  species  secondary  to  the  needs  of  nitrogen  and 
acid  extrusion  (see  above).  In  fact  we  have  found  that  the  marine  hagfish  also 
possesses  these  ionic  exchange  systems  (Evans,  1980a).  Since  hagfish  have  never 
entered  fresh  water  (Hardisty,  1979)  it  appears  that  Na+/H+  or  NH4+  exchange 
came  about  before  the  vertebrates  entered  fresh  water,  as  an  acid  and  nitrogen 
excretory  device,  rather  than  as  an  ionoregulatory  device  adaptive  to  freshwater 
existence.  The  presence  of  this  system  in  marine  species  is  therefore  an  indication 
of  an  ancient  marine  invention  rather  than  a  hold-over  from  a  former  existence  in 
fresh  water,  as  was  formerly  proposed  (Evans,  1975). 

Intact-animal  studies  have  advanced  our  knowledge  of  fish  branchial  ion  trans- 
port systems  considerably  in  the  past  20  years,  but  the  limitations  on  the  manip- 
ulation of  intact  animals  has  restricted  the  approaches  to  specific  questions  with 
somewhat  limited  answers.  While  the  use  of  intact  animals  ensures  (in  theory)  that 
proper  perfusion  and  irrigation  of  the  branchial  epithelium  is  taking  place,  and  that 
neural  and  hormonal  inputs  are  present,  it  also  ensures  that  substantial  alterations 
in  blood  ionic  components  cannot  be  made,  and  that  injection  of  known  ionic 
transport  inhibitors  may  induce  secondary  changes  via,  for  instance,  cardiovascular 
changes.  Moreover,  it  does  not  allow  one  to  separate  transport  steps  at  the  baso- 
lateral vs  apical  borders  of  the  transporting  cells.  In  addition,  the  specter  of  stress 
with  concomitant  neuroendocrine  changes  is  always  present.  For  example,  our  find- 
ing that  in  both  a  marine  teleost  and  marine  elasmobranch  replacement  of  the 
external  sea  water  with  Na+-free  artificial  sea  water  (choline  as  the  impermeant 
cation)  resulted  in  cessation  of  net  extrusion  of  FT  and  apparent  extrusion  of  base 
leads  us  to  believe  that  branchial  C1~/HCO3  exchange  may  be  present,  but  usually 
"hidden"  behind  Na+/FT  exchange,  especially  under  hypercapnic  conditions 
(Evans,  1982b).  We  tested  for  C1~/HCO3~  exchange  by  injecting  a  bicarbonate 
load  into  both  species  with  the  expectation  that  we  could  stimulate  net  base  ex- 
cretion. However,  in  both  cases  (Evans,  unpublished  observations)  we  found  that 


114  DAVID  H.  EVANS  ET  AL. 

injection  of  a  base  load  stimulated  net  H+  extrusion  rather  than  net  base  extrusion, 
secondary,  presumably,  to  a  stress  response.  In  fact  injection  of  only  Ringer's 
solution  results  in  a  net  efflux  of  H+.  Thus,  the  stress  response  (despite  the  use  of 
anesthetic)  complicates  an  investigation  of  Cr/HCO3~  exchange  in  intact  animals. 
Because  of  these  problems,  various  in  vitro  approaches  have  been  made  to  the 
study  of  fish  gill  transport  in  the  past  few  years.  We  will  start  with  perfusion  of 
the  head  end  since  this  technique  was  actually  orginated  in  the  1930's. 

THE  ISOLATED,  PERFUSED  HEAD  PREPARATION 

Keys  (193 la)  was  the  first  to  describe  a  fish  preparation  in  which  both  the 
serosal  and  mucosal  solutions  bathing  the  branchial  epithelium  could  be  controlled. 
In  this  so-called  "heart-gill"  preparation  (utilizing  the  eel,  Anguilla  anguilla)  te- 
leost  Ringer's  solution  was  perfused  into  the  hepatic  vein,  and  was  pumped  via  the 
intact,  beating  heart  to  the  gills.  External  irrigation  of  the  gills  with  a  small  volume 
of  fresh  water  was  accomplished  by  pumping  the  water  through  a  tube  inserted 
into  the  mouth  of  the  animal.  Utilizing  this  preparation,  the  first  in  vitro  experi- 
ments on  branchial  hemodynamic  and  active  chloride  transport  mechanisms  were 
described  (Keys,  1931a,b;  Bateman  and  Keys,  1932;  Keys  and  Bateman,  1932; 
Keys  and  Wilmer,  1932).  Thirty  years  later,  the  advent  of  isotopic  tracers  allowed 
a  revitalization  of  the  heart-gill  preparation  to  attempt  to  define  more  clearly  the 
NaCl  movements  across  the  gills  (Tosteson  et  al.,  1962;  Kirschner,  1969).  The 
latter  study  modified  the  "heart-gill"  preparation  of  the  eel  so  that  Ringer's  was 
perfused  into  the  ventral  aorta  via  a  pulsatile  pump,  thereby  bypassing  the  heart. 
These  pump-perfused  gills  appeared  to  be  much  more  permeable  to  Na+  than  the 
heart-gill  or  in  vivo  systems  also  tested  (Kirschner,  1969).  By  decreasing  perfusate 
temperature,  deterioration  of  ionic  fluxes  was  reduced,  but  gill  resistance  still  in- 
creased. 

The  utilization  of  the  "heart-gill"  preparation  had  one  major  undesirable  at- 
tribute: the  direct  effects  of  various  hemodynamic  agents  on  the  branchial  vascular 
(e.g.  epinephrine)  could  not  be  separated  from  their  effects  on  the  heart  itself.  The 
isolated,  perfused  head  preparation  (IPHP)  of  the  trout,  Salmo  gairdneri,  was 
developed  by  Payan  and  Matty  (1975)  and  appeared  to  be  much  more  viable  as 
a  tool  for  the  study  of  osmoregulatory  (and  hemodynamic)  parameters  of  the  gills. 

Briefly,  the  IPHP  is  prepared  by  decapitation  of  the  fish  posterior  to  the 
opercular  openings  after  heparinizing  and  anesthetizing  the  animal.  Cannulas  are 
inserted  into  the  ventral  aorta  proximal  to  the  heart,  and  into  the  mouth.  The 
preparation  is  then  placed  in  a  chamber  which  allowed  the  separation  of  irrigation 
fluid  pumped  over  the  gills  from  the  efferent  perfusate  draining  from  the  dorsal 
aorta  and  the  open  body  cavity.  Perfusion  is  accomplished  either  gravimetrically 
or  by  a  peristaltic  pump.  Afferent  flow  rate  or  perfusion  pressure  is  measured  via 
a  drop  counter  or  pressure  transducer  connected  to  the  perfusion  line.  In  some 
preparations  the  dorsal  aorta  is  cannulated,  thus  allowing  the  partitioning  of  the 
efferent  perfusate  into  dorsal  arterial  and  "venous"  components  (Girard  and  Payan, 
1976;  Claiborne  and  Evans,  1980). 

This  partitioning  of  respiratory  and  venous  flows  is  possible  since  the  blood 
leaving  the  respiratory  lamellae  in  the  gill  may  return  via  efferent  filamental  and 
branchial  arteries  to  the  dorsal  aorta  or  be  channelled  through  contractile  anas- 
tomoses between  the  efferent  filamental  artery  and  the  central  venous  sinus  of  the 
filament  to  the  venous  circulation  (Fig.  3).  In  some  species  prelamellar  anastomoses 
are  also  found  (Boland  and  Olson,  1979)  between  the  afferent  filamental  artery 
and  the  central  vein  of  the  filament,  but  these  anastomoses  are  smaller  and  less 


FISH  GILL  IONIC  TRANSPORT 


115 


FIGURE  3.  Filamental  circulation  of  the  gill  of  the  snapper,  Lutjanus  gresius.  Blood  flows  distally 
in  the  filament  in  the  afferent  filamental  artery  (afa)  which  leads  to  the  respiratory  lamellae  (rl)  via  the 
lamellar  arterioles  (la).  Blood  leaves  the  lamellae  in  the  efferent  filamental  artery  (efa).  Regular  anas- 
tomoses between  the  efferent  filamental  artery  and  the  central  venous  sinus  (cvs)  supply  the  extensive 
venous  network  surrounding  the  filamental  cartilage  (C).  Companion  nutritive  vessels  (cc)  overlay  both 
the  afferent  and  efferent  filamental  arteries  and  connect  to  the  central  venous  network  via  irregular 
anastomoses. 


1  1 6  DAVID  H.  EVANS  ET  AL. 

numerous  than  the  postlamellar  connections,  and  for  that  reason  do  not  appear  to 
form  an  effective  bypass  circuit  around  the  lamellae  (Farrell,  1980). 

Since  its  inception,  the  IPHP  has  been  used  in  a  variety  of  investigations  of  gill 
hemodynamics  (see  Claiborne  and  Evans,  1980)  and  even  brain  metabolism  (Cal- 
lard  et  al.,  1981).  We  need  concern  ourselves  only  with  the  ionic  transport  studies. 

Girard  and  Payan  (1980)  have  recently  reviewed  their  studies  on  the  IPHP  of 
the  rainbow  trout,  Salmo  gairdneri,  which  have  demonstrated  that  the  head  is 
capable  of  carrying  out  Na+/NH4+  exchange,  both  in  fresh  water  (Payan,  1978) 
and  in  sea  water  (Payan  and  Girard,  1978).  Unfortunately,  no  data  have  been 
published  on  the  coupling  of  Na+  influx  to  H+  efflux  or  Cl~  influx  to  HCO3  efflux, 
despite  the  fact  that  both  exchange  systems  have  been  described  in  the  intact  trout 
(Kerstetter  et  al.,  1970;  Kerstetter  and  Kirschner,  1972).  It  is  interesting  to  note 
that  while  Payan  (1978)  demonstrated  a  1:1  stoichiometry  for  Na+  and  influx  vs 
NH4+  efflux,  a  considerable  ammonia  efflux  (approximately  70%)  continued  in  the 
absence  of  Na+  in  the  external  bath,  indicating  clearly  that  the  majority  of  the 
ammonia  efflux  is  not  coupled  to  external  Na+.  These  data  corroborate  our  finding 
(Evans,  1977)  that  50%  or  less  of  the  ammonia  efflux  from  intact  marine  species 
is  dependent  upon  seawater  Na+.  By  examining  the  partitioning  of  the  postlamellar 
(see  Laurent  and  Dunel,  1980  for  a  review  of  fish  gill  morphology)  perfusate  flows 
into  dorsal  aorta  vs  "venous"  flows,  Girard  and  Payan  (1977a)  were  able  to  dem- 
onstrate that  all  of  the  Na+  and  Cl  influx  was  across  the  lamellar  epithelium, 
contrary  to  the  situation  in  the  perfused  head  of  the  seawater-adapted  trout  where 
a  significant  portion  of  the  influx  is  across  the  filamental  surfaces,  presumably  inter- 
lamellar  (Girard  and  Payan,  1977b).  Since  this  lamellar  Na+  influx  in  the  fresh- 
water-adapted head  displays  the  characteristics  of  Na+/NH4+  exchange  (i.e.  is 
sensitive  to  perfusate  NH4+  concentrations;  Payan,  1978)  it  appears  that  the  active 
transport  step  for  at  least  freshwater  Na+  balance  is  in  the  lamellar  epithelium, 
rather  than  the  so-called  "chloride  cells"  of  the  filamental  epithelium.  This  is  the 
first,  and  only,  evidence  that  we  have  that  ionic  extraction  by  freshwater  fish  may 
actually  not  involve  the  "Cl  cells,"  which  are  generally  thought  to  be  the  sites  of 
active  salt  transport  in  both  freshwater  and  seawater  fish  (see  below).  It  is  important 
to  note  that  the  influxes  of  both  Na+  and  Cl~  displayed  by  the  perfused  trout  head 
were  only  20-30%  of  those  found  in  vivo  (Girard  and  Payan,  1977a);  however,  10~! 
M  epinephrine  stimulated  Na+  influx  to  some  130%  of  in  vivo,  while  the  Cl"  influx 
remained  unchanged  (Girard  and  Payan,  1977a). 

The  IPHP  has  also  been  utilized  to  examine  more  carefully  the  cellular  local- 
ization of  the  Na+/NH4+  exchange  mechanism.  Payan  et  al.  (1975)  demonstrated 
that  addition  of  ouabain  inhibited  both  Na+  influx  and  ammonia  efflux  from  per- 
fused freshwater  trout  heads.  However,  Payan  (1978)  proposed  that  this  inhibition 
was  secondary  to  a  primary,  apical  Na+/NH4+  exchange  which  was  sensitive  to 
intracellular  Na+  concentrations  (which  were  maintained  by  basolateral  Na+-K+ 
ATPase).  This  model  was  based  upon  his  finding  that  acetazolamide  added  to  the 
perfusate  inhibited  ammonia  clearance,  as  did  amiloride  added  to  the  irrigation 
fluid  (fresh  water).  More  critically,  he  found  that  reducing  the  NH3  concentration 
of  the  perfusate  by  approximately  10-fold  (by  reducing  the  pH  by  1  pH  unit) 
inhibited  the  excretion  of  ammonia  by  some  85%.  It  also  inhibited  sodium  influx 
by  about  60%  (Payan,  1978).  He  therefore  proposed  that  ammonia  entered  the  cell 
as  NH3,  was  proton ated  via  the  hydration  of  CO2  via  carbonic  anhydrase,  and  was 
excreted  at  the  apical  surface  in  exchange  for  Na+  in  the  fresh  water — the  model 
first  proposed  by  Maer/  and  Garcia  Romeu  in  1964  for  intact  fish  (see  above).  We 


FISH  GILL  IONIC  TRANSPORT  1  1  7 

have  recently  approached  the  same  problem  with  the  IPHP  of  two  marine  teleost 
fishes  (Myoxocephalus  octodecimspinosus,  the  longhorned  sculpin;  and  Opsanus 
beta,  the  gulf  toadfish)  and  found  that  increasing  the  perfusate  NH3  concentration 
(by  increasing  the  pH)  did  not  stimulate  ammonia  efflux;  however,  increasing  only 
the  NH4+  concentration  (by  increasing  the  ammonia  concentration,  while  reducing 
the  pH)  stimulated  the  ammonia  efflux  significantly  (Goldstein  et  al.,  1982).  Since 
only  approximately  50%  of  the  ammonia  efflux  is  coupled  to  Na+  in  intact  marine 
fishes  (see  above),  it  appears  that  a  significant  component  of  ammonia  efflux  from 
at  least  marine  fish  gills  is  via  diffusion  of  NH4+  across  the  branchial  epithelium. 
This  may  be  via  leaky  "tight  junctions"  since  the  marine  teleost  gill  has  been  shown 
to  be  quite  leaky  to  cations  and  even  large  organic  molecules  (Karnaky,  1980).  The 
proposition  that  NH4+  can  diffuse  across  the  marine  teleost  gill  is  supported  by 
our  earlier  finding  that  addition  of  200  mM  NH4C1  solutions  to  Na-free  artificial 
sea  water  depolarized  the  TEP  across  the  intact  toadfish  to  the  same  extent  as  200 
mM  NaCl  (Evans,  1977).  It  is  important  to  note  that  the  ammonia  efflux  from  the 
IPHP  of  both  O.  beta  and  M.  octodecimspinosus  is  close  to  that  found  in  vivo 
(Goldstein  et  al.,  1982).  We  have  found  that  ouabain  added  to  the  Ringer's  solution 
(containing  1  mM  NH4C1)  perfusing  the  IPHP  of  O.  beta  inhibited  ammonia  efflux 
by  some  50%.  This  could  have  been  an  indirect  effect  (as  proposed  by  Payan,  1978); 
however,  we  have  also  found  that  addition  of  K+  to  the  perfusate  inhibited  ammonia 
efflux,  indicating  a  direct  interaction  at  the  basolateral  border.  In  addition,  we 
found  that  neither  ouabain  nor  K+  produced  hemodynamic  effects  sufficient  to 
account  for  the  observed  inhibition  of  ammonia  efflux  (Claiborne  et  al.,  1982).  We 
conclude  that,  at  least  in  this  species,  Na+/NH4+  exchange  is  basolateral,  rather 
than  apical,  and  running  through  the  Na+-K+  activated  ATPase.  The  NH4+  sen- 
sitivity of  this  enzyme  is  well  documented  (see  below). 

Since  intact  marine  teleosts  and  elasmobranches  have  been  shown  to  excrete 
H+  in  exchange  for  Na4  (Evans  et  al.,  1979;  Evans,  1982b)  it  would  be  of  great 
interest  to  use  an  IPHP  to  examine  this  system  in  greater  detail. 

Girard  (1976)  used  the  IPHP  of  the  seawater-adapted  trout  to  examine  various 
aspects  of  the  extrusion  of  Na+  and  Cr.  He  found  that  the  effluxes  of  Na+  is  near 
to  that  measured  in  vivo  and  that  effluxes  of  both  Na+  and  Cl"  were  stimulated 
by  addition  of  K+  to  the  external  medium;  unfortunately  he  did  not  report  TEPs 
so  that  one  could  separate  chemical  vs  electrical  coupling.  Claiborne  and  Evans 
(1981)  have  recently  shown  that  the  IPHP  of  M.  octodecimspinosus  maintains  a 
Na+  efflux  near  in  vivo  levels,  but  a  Cl"  efflux  significantly  below  that  found  in  the 
intact  fish.  The  efflux  of  neither  ion  is  affected  by  large  alterations  in  the  irrigation 
rate,  but  changes  in  perfusion  rate  (and  therefore  pressure)  produce  significant 
alteration  in  the  Na+  efflux,  with  no  effect  on  the  Cl"  efflux.  This  argues  for  separate 
pathways  for  the  bulk  of  the  Na+  vs  Cl"  efflux  which  supports  the  extrusion  model 
of  Silva  et  al.  (1977;  see  above),  but  does  not  support  the  recent  porposition  (Sargent 
et  al.,  1 978;  Kelly  et  al.,  1981)  that  NaCl  is  forced  across  the  leaky  "tight  junctions" 
of  the  branchial  epithelium  by  arterial  blood  presure. 

Various  direct  tests  of  the  "Silva  model"  are  theoretically  possible  with  the 
IPHP.  Since  the  perfusate  can  be  manipulated  it  would  be  of  great  interest  to  test 
the  sensitivity  of  the  Cl  efflux  to  removal  of  Na+  from  the  perfusate.  This  would 
be  the  most  direct  test  of  the  proposed  co-transport  of  Na+  and  Cl"  which  is  the 
core  of  this  model.  Unfortunately,  the  branchial  vasculature  of  at  least  M.  octo- 
decimspinosus is  quite  sensitive  to  the  choline  used  to  replace  the  perfusate  Na+ 
and  subsequent  large  increases  in  afferent  pressure  and  Cl"  efflux  obscure  any 


118  DAVID  H.  EVANS  ET  AL. 

changes  in  Cl~  efflux  which  may  have  been  produced  by  the  lack  of  Na+  (Claiborne 
and  Evans,  unpublished). 

Kelly  et  al.  (1981)  have  recently  found  that  10~4  M  ouabain  inhibits  both  the 
Na+  and  Cl~  efflux  from  the  IPHP  of  the  eel  (Anguilla  anguilla}  by  some  30-40% 
with  no  effect  on  the  afferent  perfusion  pressure,  or  the  efflux  of  tritiated  water. 
Thus,  in  these  experiments,  one  can  be  rather  certain  that  the  effect  of  ouabain 
was  a  direct  one  on  some  component  of  the  transport  system,  rather  than  an  indirect 
effect  through  hemodynamic  changes. 

To  date,  no  report  of  a  transepithelial  potential  measured  across  the  gills  of  an 
IPHP  has  appeared  in  the  literature.  TEP  changes  across  the  branchial  epithelium 
must  be  monitored  concurrent  with  ion  substitution  or  drug  inhibition  experiments 
(see  above).  Recently,  we  have  found  it  possible  to  measure  the  TEP  across  the 
gills  of  the  IPHP  of  M.  octodecimspinosus  in  sea  water.  We  found  that  the  IPHP 
TEP  was  similar  to  that  measured  in  vivo.  Substitution  of  Na+  or  Cl~  with  the 
appropriate  impermeant  ion  in  the  external  sea  water  resulted  in  large  depolariza- 
tions when  Na+  was  replaced,  but  no  alterations  were  observed  after  Cl~  substi- 
tutions (Claiborne  and  Evans,  1981).  These  responses,  observed  both  in  vivo  and 
in  vitro,  indicate  that  the  gills  of  the  IPHP  (and  the  sculpin  in  vivo)  are  more 
permeable  to  Na+  than  to  Cl~,  as  shown  in  many  other  teleosts  which  possess  a 
positive  TEP  (Evans,  1979). 

While  the  IPHP  enables  the  investigator  to  ask  questions  impossible  using  intact 
systems,  it  still  presents  some  limitations.  The  majority  of  the  studies  of  ion  trans- 
port by  the  IPHP  have  utilized  the  trout  head,  which  suffers  from  rather  serious 
hemodynamic  degradation  in  a  short  period  of  time.  Girard  (1976)  found  that  the 
gill  resistance  increased  by  some  5-fold  within  30  minutes  and  Wood  (1974)  found 
that  relatively  linear  and  stable  pressure  vs  flow  relationships  were  only  possible 
if  post-branchial  efferent  pressures  were  maintained  by  a  column  of  irrigation 
solutions.  To  delay  the  hemodynamic  degradation  of  the  trout  head,  epinephrine 
has  sometimes  been  added  to  the  perfusate  (Payan,  1978).  However,  it  is  clear  that 
this  hormone  stimulates  Na+  uptake  in  fresh  water  and  inhibits  it  in  sea  water 
(Girard,  1976;  Payan,  1978;  Shuttleworth,  1978).  This  hemodynamic  degradation 
of  the  IPHP  may  be  species  specific  since  we  have  recently  found  that  IPHPs  of 
the  sculpin,  toadfish,  and  shark  "pup"  (Squalus  acanthias)  can  maintain  relatively 
consistant  gill  resistances  for  3-8  hours  (Claiborne  and  Evans,  1980;  Oduleye  et 
al.,  unpublished  results;  Evans  and  Claiborne,  1982).  In  all  three  species  the  afferent 
pressures  are  at  in  vivo  levels  when  the  perfusion  rate  is  in  the  same  range  as  the 
in  vivo  cardiac  output,  despite  the  fact  that  postbranchial  efferent  resistances  are 
near  zero.  It  is  obvious  that  other  species  should  be  examined. 

Importantly,  most  of  the  IPHP  studied  to  date  maintain  Na+  and/or  Cl~  fluxes 
significantly  below  in  vivo  levels  (see  above).  In  fact,  in  a  recent  study  using  the 
IPHP  of  A.  anguilla  the  measured  Na+  and  Cl  fluxes  were  only  10%  of  the  fluxes 
measured  in  vivo  (compare  Kelly  et  al.,  1981,  with  Epstein  et  al.,  1973).  Whether 
the  reduced  effluxes  found  in  some  species  are  secondary  to  incomplete  perfusion 
of  the  branchial  vasculature  or  lack  of  stimulatory  hormones  normally  found  in 
vivo  remains  to  be  determined. 

Probably  the  most  important,  and  least  often  controlled,  parameter  of  the  IPHP 
is  the  ratio  of  the  perfusate  inflow  to  outflow.  Most  authors  do  not  note  this  com- 
parison which  is  a  direct  measure  of  the  structural/hemodynamic  integrity  of  the 
system.  It  should  be  obvious  that  even  slight  leakage  of  the  perfusate  either  into 
the  external  medium  or  the  head  tissues  will  produce  quite  spurious  determination 


FISH  GILL  IONIC  TRANSPORT  119 

of  ion  flux  rates.  These  leak  pathways  may  not  affect  active  pathways,  but  they 
may  obscure  the  latter's  importance  or  even  presence  in  the  total  unidirectional 
flux  as  determined  with  radioisotopes.  Losses  of  up  to  30%  of  the  perfusate  during 
its  transit  of  the  gills  has  been  reported  by  some  investigators  in  personal  com- 
munications. Again  this  may  be  species  specific  because  we  have  found  that  the 
sculpin,  toadfish,  and  dogfish  shark  "pup"  maintain  inflow:outflow  ratios  of  ap- 
proximately 1.0  (Claiborne  and  Evans,  1980;  Oduleye  et  al.,  unpublished  results; 
Evans  and  Claiborne,  1982).  In  summary,  present  data  indicate  that  the  isolated, 
perfused  head  preparation  may  allow  a  more  critical  dissection  of  the  mechanisms 
of  NaCl  transport  by  the  fish  branchial  epithelium  than  is  possible  with  in  vivo 
studies.  It  is  important  to  note  that  in  most  instances  published  IPHP  studies  have 
corroborated  the  findings  of  earlier  studies  using  intact  animals,  despite  the  fact 
that  many  of  the  preparations  (especially  those  utilizing  the  trout  head)  display 
significant  degeneration  of  the  hemodynamics  of  the  branchial  vasculature.  It  is 
clear  that  more  species  need  to  be  investigated  and  that  greater  attention  be  paid 
to  the  ratio  of  the  inflows:outflows  and  the  TEPs  maintained  by  the  IPHP. 

THE  ISOLATED,  PERFUSED  GILL  PREPARATION 

An  alternative  to  the  perfused  head  is  the  isolated,  perfused  gill,  which  has  been 
used  rather  extensively  in  the  past  1 5  years.  Although  methods  vary  slightly,  gen- 
erally isolated  gills  are  prepared  by  initial  perfusion  of  an  anesthetized  animal  with 
Ringer's  solution.  When  filaments  are  free  of  blood,  individual  arches  are  selected 
and  removed.  The  afferent  and  efferent  branchial  arteries  are  cannulated,  and  the 
arch  is  placed  in  a  well-stirred  external  bath.  In  early  work  a  constant  pressure 
reservoir  provided  afferent  pressure,  but  more  recently  pulsatile  flow  generated  by 
a  pump  has  been  employed.  Efferent  pressure  is  set  by  the  height  of  the  efferent 
cannula  above  the  preparation. 

Like  the  perfused  head,  the  perfused  gill  preparation  has  been  used  extensively 
to  investigate  the  hemodynamics  of  branchial  circulation,  but  rather  little  to  study 
gill  ion  transport.  To  a  considerable  extent  this  is  apparently  due  to  the  isolated 
gill's  ability  to  maintain  reasonable  hemodynamics  (e.g.  Bergman  et  al.,  1974; 
Holbert  et  al.,  1979)  but  inability  to  maintain  proper  irrigation.  Unfortunately, 
even  vigorous  stirring  of  the  irrigation  bath  apparently  does  not  mimic  the  irrigation 
patterns  found  in  the  intact  animal,  or  the  perfused  head.  For  example,  Shuttleworth 
and  Freeman  ( 1974)  described  Na+  and  Cl  effluxes  from  the  perfused  eel  (Anguilla 
dieffenbachii}  gills  that  were  only  10-15%  of  those  found  in  the  intact  fish,  and 
Farmer  and  Evans  ( 1981 )  have  recently  found  that  the  efflux  of  Cl~  from  perfused 
pinfish  (Lagodon  rhomboides}  gills  is  45%  that  of  the  intact  fish.  Nevertheless,  the 
perfused  gill  has  provided  us  with  some  information  unavailable  with  other  tech- 
niques. Shuttleworth  et  al.  (1974)  demonstrated  that  the  TEP  across  the  perfused 
marine  flounder  (Platichthyes  flesus}  gill  was  approximately  7  mV  inside  positive 
when  the  gill  was  perfused  and  irrigated  with  Ringer's  solution.  Addition  of  ouabain 
inhibited  the  TEP,  indicating  that  salt  extrusion  was  electrogenic,  and  that  Na+- 
K+  activated  ATPase  played  an  important  role.  The  finding  of  a  substantial  TEP 
when  no  chemical  gradients  existed  across  the  gill  epithelium  demonstrated  that 
the  TEP  across  intact  marine  fish  was  probably  a  combination  of  electrogenic 
transport  and  differential  ionic  permeabilities.  Studies  with  intact  marine  fish  had 
suggested  that  the  TEP  was  primarily  the  result  of  a  much  higher  cation  than 
anion  permeability  (Potts  and  Eddy,  1973;  Kirschner  et  al.,  1974).  More  recent 
studies  have  lent  support  for  the  "Silva  model"  for  coupled  Na+  and  Cl~  transport 


120  DAVID  H.  EVANS  ET  AL. 

by  the  gill  epithelium.  Farmer  and  Evans  (1981)  have  shown  that  the  Cl  efflux 
from  the  perfused  pinfish  gill  is  inhibited  by  removal  of  Na+  from  the  perfusate, 
or  addition  of  furosemide.  Furosemide  has  been  found  to  inhibit  coupled  Na+  and 
Cl~  transport  in  a  wide  variety  of  epithelial  tissues  (Frizell  et  al.,  1979). 

The  perfused  gill  has  been  utilized  to  examine  salt  uptake  by  freshwater  fish. 
Richards  and  Fromm  (1970)  found  that  addition  of  ouabain  to  the  Ringer's  solution 
perfusing  the  isolated  trout  gill  inhibited  the  uptake  of  Na+  and  Shuttleworth  and 
Freeman  (1974)  found  that  removal  of  K+  from  the  perfusate  inhibited  Na+  uptake 
by  the  eel  gill.  Both  studies  support  the  conclusion  that  basolateral  Na+/K+  ex- 
change (mediated  via  Na+-K+  activated  ATPase)  plays  a  role  in  Na+  uptake  in 
fresh  water. 

THE  ISOLATED  OPERCULAR  EPITHELIUM 

An  extensive  literature  indicates  that  the  mitochondria-rich  "chloride  cell"  of 
the  fish  gill  epithelium  plays  an  important  role  in  osmoregulation  (for  an  extensive 
review  see  The  Biology  of  the  Chloride  Cell:  Jean  Maetz  Memorial  Symposium, 
American  Journal  of  Physiology  238:  R141-R276,  1980).  Quite  recently  a  tech- 
nique has  been  developed  which  has  enabled  a  much  more  direct  study  of  the 
biophysics  of  ion  transport  across  this  cell  than  has  been  possible  with  intact  fish, 
or  isolated  heads  or  gills. 

Burns  and  Copeland  (1950)  demonstrated  that  "chloride  cells"  are  widely  dis- 
tributed throughout  the  head  region  of  the  killifish,  Fundulus  heteroclitus,  but  it 
was  not  until  1977  that  it  was  shown  that  the  opercular  epithelium  of  this  species 
possesses  a  cellular  population  which  is  50-70%  "chloride  cells"  whose  cytology 
and  ultrastructure  is  identical  to  the  "chloride  cells"  in  the  gill  epithelium  (Fig. 
4)  (Karnaky  et  al.,  1976;  Karnaky  and  Kinter,  1977).  Thus,  the  opercular  epithe- 
lium presented  the  unique  opportunity  to  investigate  the  function  of  "chloride  cells" 
on  a  flat  epithelium,  rather  than  on  the  extremely  complex  branchial  epithelium. 
A  flat  epithelium  can  be  dissected  free  and  mounted  in  an  "Ussing  Chamber" 
which  enables  a  strict  thermodynamic  approach  to  the  electrical  and  chemical 
events  of  ion  transport.  In  this  way  one  can  carefully  control  the  ionic  composition 
of  both  serosal  and  mucosal  solutions  bathing  the  tissue  and  measure  net  movements 
of  ions  quite  accurately.  In  addition,  any  spontaneously  generated  electrical  po- 
tentials can  be  measured  and  nulled  (to  quantify  the  short-circuit  current),  and 
resistances  can  be  calculated.  Since  the  original  description  of  the  opercular  epi- 
thelium of  F.  heteroclitus,  similar,  "chloride  cell"-rich  tissues  have  been  found  in 
the  operculum  of  F.  grandis  (Krasny  and  Evans,  1980)  and  Sarotherodon  mos- 
sambicus  (Foskett  et  al.,  1979)  as  well  as  the  jaw  epithelium  of  Gillichthys  mir- 
abilis  (Marshall  and  Bern,  1980). 

When  the  isolated  opercular  epithelium  from  seawater-adapted  killifish  is 
bathed  bilaterally  with  a  Ringer's  solution  having  an  ionic  composition  similar  to 
F.  heteroclitus  plasma,  a  potential  difference  oriented  serosa  (blood)  positive  is 
generated  (Degnan  et  al.,  1977;  Karnaky  et  al.,  1977).  Subsequent  isotopic  flux 
studies  (Table  I)  showed  that  this  potential  difference  was  the  result  of  the  net 
transport  of  Cl  outwards  across  the  tissue,  i.e.  blood  side  to  seawater  side;  there 
was  no  net  transport  of  Na+  across  the  epithelium  (Degnan  et  al.,  1977;  Karnaky 
et  al.,  1977).  These  were  the  first  unequivocal  studies  showing  that  killifish  maintain 
ionic  homeostasis  in  sea  water  by  actively  extruding  chloride  into  the  external 
milieu.  Equivalancy  between  the  short-circuit  current  and  net  Cl~  secretion  has 
also  been  observed  in  the  "chloride  cell"  containing  opercular  epithelia  of  F.  grandis 


FISH  GILL  IONIC  TRANSPORT 


121 


Apical  crypt 


Pavement  cells 


Fuzzy  coat 


Mucous  cell 


Glycogen 
granules 


Non 

differentiated 
cells 


Pavement  cells 


Mucous  cell 


Area  enlarged 


Apical  crypts 


Chloride 

• 

cells 


i !  ^ffelr  \«\ 


Connective 
Basal  lamina          tissue 


Fibrocyte 


Capillary 


FIGURE  4.  Schematic  of  the  ultrastructure  of  a  "chloride  cell"  (upper)  and  opercular  epithelium 
(lower)  from  the  opercular  epithelium  from  Fundulus  heteroclitus.  In  this  tissue  50-70%  of  the  cellular 
population  is  represented  by  "chloride  cells"  whose  cytology  is  identical  to  that  described  for  the  branchial 
epithelium  of  teleosts.  Reproduced  with  kind  permission  from  Degnan  et  al.  ( 1977).  See  text  for  details 
of  the  physiology  of  this  opercular  tissue.  Scale  is  20  Mm. 


122  DAVID  H.  EVANS  ET  AL. 

TABLE  I 

Isotopic  fluxes  and  electrical  properties  across  the  short-circuited  opercular  epithelia  of  seawater- 
adapted  Fundulus  heteroclitus  gassed  with  95%  oxygen,  5%  carbon  dioxide. 

Efflux  Influx  Net  Flux  SCC  PD 


Cl 

Na 

7.23  ±  2.13 
2.63  ±  0.45 

2.86  ±  1.13 
2.95  ±  0.26 

4.46  ±  1.09/119.6  ±  29.3 
-0.32  ±  0.62/-8.6  ±  16.5 

119.2  ±  22.9 
74.4  ±  10.3 

12.6  ± 
10.1  ± 

1.2 
1.5 

N  for  fluxes  is  8,  N  for  electrical  properties  is  16.  Fluxes  in  ^Eq-cm  2-h  ',  net  fluxes  in 
crrr2-h  '/^A-crrT2.  Short  circuit  current  (SCC)  in  nM-cm~2  and  potential  difference  (PD)  in  mV, 
serosa  relative  to  mucosa.  Data  from  Degnan  el  at.  (1977).  Note  that  the  SCC  is  identical  to  the  net 
influx  of  Cl  with  no  net  movements  of  Na. 

(Krasny,  1981)  and  Sarotherodon  mossambicus  (Foskett  el  al.,  1979)  and  jaw 
epithelium  of  Gillichthys  mirabilis  (Marshall  and  Bern,  1980). 

The  transport  mechanisms  in  "chloride  cells"  for  chloride  appears  very  similar 
to  that  found  in  most  chloride-transporting  epithelial  types  (Frizzell  et  al.,  1979; 
Frizzell  and  Duffey,  1980).  Namely,  Cl~  efflux  is  dependent  upon  the  presence  of 
Na+  in  the  serosal  medium  (Degnan  and  Zadunaisky,  1980a,  1981;  Mayer-Gostan 
and  Maetz,  1980)  and  is  blocked  by  the  transport  inhibitors  furosemide  or  ouabain 
on  the  serosal  side  (Degnan  et  al.,  1977;  Karnaky  et  al.,  1977;  Mayer-Gostan  and 
Maetz,  1980). 

Studies  utilizing  the  short-circuit  current  techniques  made  in  conjunction  with 
fluorescence  microscopy  techniques  have  provided  direct  evidence  that  the  "chloride 
cell"  is  the  "active"  ionocyte  involved  in  seawater  teleost  osmoregulation.  DASPMI, 
a  low  toxicity,  specific  fluorescent  stain  for  mitochondria  in  living  cells  (Bereiter- 
Hahn,  1976),  has  been  used  to  stain  "chloride  cells"  in  the  opercular  epithelium 
of  F.  heteroclitus  (Zadunaisky,  1979).  Studies  using  this  dye  in  the  opercular 
epithelium  of  F.  heteroclitus  (Karnaky  et  al.,  1979)  and  the  jaw  skin  epithelium 
of  Gillichthys  mirabilis  (Marshall  and  Nishioka,  1980)  have  shown  a  linear  cor- 
relation between  "chloride  cell"  density  and  the  magnitude  of  the  short-circuit 
current.  Similarly,  Foskett  et  al.  (1979)  have  shown  that  the  increase  in  "chloride 
cell"  density  and  size  is  correlated  to  the  development  of  a  short-circuit  current 
in  the  opercular  epithelium  of  Sarotherodon  when  the  fish  is  acclimated  to  sea 
water.  More  recently,  Foskett  and  Scheffey  (1982)  have  found,  using  a  vibrating 
probe  technique,  that  current  generated  by  the  short-circuited  opercular  epithelium 
is  directly  over  the  "chloride  cells".  This  is  certainly  the  most  definitive  demon- 
stration that  the  "chloride  cells"  are  the  site  of  electrogenic  Cl~  transport  across 
the  fish  branchial  epithelium. 

By  studying  the  voltage  dependency  of  the  unidirectional  flux  of  an  ion  across 
an  epithelium  one  can  predict  the  nature  (conductive  versus  electroneutral)  and 
the  pathway  (cellular  versus  paracellular)  of  ion  flow  (Frizzell  and  Schultz,  1972; 
Mandel  and  Curran,  1972).  Results  from  studies  made  in  opercular  epithelia  of 
F.  heteroclitus  (Degnan  and  Zadunaisky,  1980b)  and  F.  grandis  (Krasny,  1981) 
indicated  that  there  were  no  significant  differences  between  the  predicted  and  mea- 
sured fluxes  for  either  the  efflux  or  influx  of  Na+,  thus  allowing  the  conclusion  that 
the  Na+  fluxes  in  opercular  epithelia  are  passive  and  traverse  only  one  rate-limiting 
barrier.  This  rate-limiting  barrier  presumably  is  represented  by  the  tight  junctional 
complex  between  neighboring  "chloride  cell"  (Sardet  et  al.,  1979;  Ernst  et  al., 
1980)  as  is  indicated  from  experiments  with  triaminopyrimidine  (TAP).  TAP, 
which  blocks  passive  cation  transport  through  the  paracellular  pathway  in  "leaky" 
epithelia  (Moreno,  1975)  reduces  the  Na+  efflux  84.1%,  while  reducing  the  total 


FISH  GILL  IONIC  TRANSPORT  1  23 

tissue  conductance  77%,  in  the  opercular  epithelium  of  F.  heteroclitus  (Degnan 
and  Zadunaisky,  1980b). 

These  results  suggesting  passive  Na+  movements  as  well  as  the  results  from 
studies  made  on  the  mechanism  of  chloride  secretion  in  the  opercular  epithelium 
have  provided  strong  direct  evidence  for  the  Silva  et  al.  (1977)  model  for  "chloride 
cell"  function  in  seawater  teleosts. 

Whereas  the  isolated  opercular  epithelium  from  seawater-adapted  teleosts  has 
been  used  to  define  the  ion  transport  properties  of  "chloride  cells,"  the  use  of  this 
preparation  in  the  study  of  freshwater  ion  regulation  is  relatively  uninvestigated. 
Although  intact  F.  heteroclitus  maintain  ionic  homeostasis  in  fresh  water  by  ex- 
tracting Na+  and  Cl~  from  the  environment  (Maetz  et  al.,  1967;  Potts  and  Evans, 
1967),  opercular  epithelia  from  freshwater-adapted  F.  heteroclitus  continue  to  se- 
crete Cl~  (Degnan  et  al.,  1977).  This  may,  in  fact,  be  due  to  autoregulation  of  the 
apical  membrane  permeability  to  Cl  induced  by  the  exposure  of  freshwater  op- 
ercular epithelia  to  a  Ringer  bathing  media  containing  142.5  mM  Cl,  i.e.  chloride 
regulates  its  own  membrane  permeability  (Ques-von  Petery  et  al.,  1978).  Evidence 
for  this  supposition  can  be  found  in  experiments  performed  on  seawater-adapted 
opercular  epithelia  where  removal  of  Cl  from  the  mucosal  bathing  media  results 
in  a  decrease  in  tissue  conductance  and  reduces  the  rate  of  Cl~  secretion  (Degnan 
and  Zadunaisky,  1980a).  On  the  other  hand,  opercular  epithelia,  isolated  from 
normally  freshwater-occurring  Sarotherodon  (Foskett  et  al.,  1979)  or  from  F.  het- 
eroclitus which  had  been  chronically  injected  with  the  "freshwater"  hormone  pro- 
lactin  (Mayer-Gostan  and  Zadunaisky,  1978),  are  characterized  by  low  short-cir- 
cuit currents  and  high  electrical  resistances.  This  might  be  expected  since:  1 )  the 
active  chloride  secretory  process  is  "turned  off;"  and  2)  the  proposed  ionic  uptake 
mechanisms  for  both  Na+  and  Cl"  in  the  branchial  epithelium  of  freshwater  teleosts 
are,  in  fact,  one  for  one  electroneutral  (electrically  silent)  exchanges:  Na+/H+  and/ 
or  NH4+  and  C1/HCO3~  (see  above). 

Although  it  has  been  suggested  that  the  Na+/H+  or  Na+/NH4+  exchanger  may 
be  located  in  the  pavement  cells  of  the  lamellae  of  the  branchial  epithelium  (Girard 
and  Payan,  1980),  these  cells  are  derived  from  the  filamental  epithelium  (Morgan, 
1974;  Laurent  and  Dunel,  1980)  and  are  identical  to  the  pavement  cells  of  the 
opercular  epithelium  as  determined  by  thin  section  electron  microscopy  (Karnaky 
and  Kinter,  1977;  Ernst  et  al.,  1980)  and  freeze-fracture  (Sardet  et  al.,  1979;  Ernst 
et  al.,  1980)  techniques.  Thus,  the  use  of  the  isolated  opercular  epithelial  prepa- 
ration with  the  pH-stat  technique  may  yield  new  and  important  information  con- 
cerning the  ionic  mechanisms  involved  in  acid-base  balance  in  both  freshwater  and 
seawater  teleosts. 

In  theory,  the  isolated  opercular  epithelium  may  provide  us  with  a  vehicle  for 
studying  intracellular  ionic  concentrations  and  basolateral  vs  apical  transport  events 
via  microelectrodes,  in  a  manner  similar  to  that  recently  used  for  a  variety  of 
transporting  epithelia  (Frizell  et  al.,  1979).  However,  the  complex  geometry  of  the 
extensive  basolateral  tubular  invaginations  results  in  a  relatively  sparse  cytoplasm 
which  may  hinder  such  determinations. 

It  is  obvious  that  the  isolated  opercular  epithelium  has  allowed  substantial 
advances  in  the  investigation  of  the  biophysics  of  NaCl  extrusion  by  a  seawater- 
acclimated  teleost.  However,  one  must  be  cautious  when  extending  these  data  to 
all  marine  teleosts,  and  especially  those  species  which  seem  to  maintain  Na+  out 
of  electrochemical  equilibrium.  In  addition,  it  remains  to  be  seen  if  it  will  be  useful 
for  the  investigation  of  other  transport  events  such  as  Na+/NH4+,  Na+/H+,  and 
Cr/HCO3  exchange. 


1  24  DAVID  H.  EVANS  ET  AL. 

ISOLATION  AND  CHARACTERIZATION  OF  TRANSPORT  ATPASES 

The  foregoing  demonstrates  the  central  role  of  Na+-K+  activated  ATPase  in 
ion  balance  and  nitrogen  excretion  by  the  teleost  branchial  epithelium.  This  subject 
has  also  been  recently  reviewed  by  Epstein  et  al.  (1980),  Karnaky  (1980),  and 
Towle  (1981 ).  The  assay  of  enzymatic  activity  primarily  in  whole  gill  homogenates 
has  been  especially  productive  in  assessing  salinity  adaptive  changes.  This  approach 
may  be  biased  by  differences  between  biochemical  techniques  (homogenization 
time,  temperature,  pH  detergents,  etc.}  of  different  laboratories,  and  changes  in 
tissue  protein  levels  which  will  bias  specific  activity  measurements  if  microsomal 
fractions  are  used.  Unfortunately,  the  biochemical  isolation  and  characterization 
of  this  presumptive  transport  enzyme  is  rather  rare.  Isolation  and  purification  is 
certainly  the  approach  which  ought  to  be  more  productive  in  defining  the  ionic 
parameters  that  this  gill  enzyme  functions  under. 

Partial  characterizations  of  branchial  Na+-K+  activated  ATPase  have  been 
published  (Kamiya  and  Utida,  1969;  Pfeiler  and  Kirschner,  1972;  Giles  and  Van- 
stone,  1976;  Ho  and  Chan,  1980),  but  the  publications  from  Sargent's  laboratory 
(Sargent  and  Thomson,  1974;  Bell  et  al.,  1977;  Bell  and  Sargent,  1979;  Sargent 
et  al.,  1980)  present  the  most  detailed  analysis  of  the  enzyme  from  the  fish  gill 
(the  Atlantic  eel,  Anguilla  anguilla).  They  have  purified  the  Na+-K+  activated 
ATPase  to  a  specific  activity  of  approximately  400  \iM ' •  mg  protein^1  •  h~',  one  to 
two  orders  of  magnitude  greater  than  that  described  by  other  authors  (see  Kir- 
schner, 1980  for  representative  data).  The  enzyme,  like  that  isolated  from  mam- 
malian kidney  and  shark  rectal  gland  (Dahl  and  Hokin,  1974;  Schwartz  et  al., 
1975)  is  phosphorylated  in  the  presence  of  Na+  and  Mg  •  ATP  to  produce  a  phos- 
phoenzyme  intermediate,  which  is  dephosphorylated  in  the  presence  of  K+.  Ouabain 
inhibits  the  dephosphorylation  step  and  other  cations,  including  NH4+,  can  sub- 
stitute for  K+  at  the  dephosphorylation  step,  with  varying  affinities.  Bell  et  al. 
(1977)  found  that  the  affinity  of  the  purified  enzyme  for  NH4+  was  slightly  less 
than  for  K+,  while  Mallery  (1979)  found  that  partially  purified  enzyme  from  O. 
beta  displayed  a  higher  affinity  for  NH4+  than  for  K+.  It  is  interesting  to  note  that 
this  species  displays  a  ouabain  and  K+-sensitive  ammonia  efflux  (Claiborne  et  al., 
1982).  Unfortunately  we  have  no  data  on  the  molecular  weight  or  subunit  structure 
of  the  fish  branchial  Na+-K+  activated  ATPase. 

We  know  even  less  about  a  putative  anionic  transport  ATPase.  Kerstetter  and 
Kirschner  (1974)  described  an  ATPase  fraction  from  trout  branchial  tissue  which 
was  stimulated  by  HCO3~  and  inhibited  by  thiocyanate.  Both  the  enzyme  and  Cl~ 
influx  was  inhibited  by  thiocynate,  which  is  especially  surprising  considering  that 
the  enzyme  was  inhibited,  rather  than  stimulated  by  the  addition  of  Cl~  to  the 
incubation  medium.  Importantly,  comparison  with  succinic  dehydrogenase  activity 
as  a  mitochondrial  marker  indicated  that  the  HCO3~-stimulated  ATPase  was  in 
both  mitochondrial  and  microsomal  fractions.  More  recently  DeRenzis  and  Bor- 
nancin  (1977)  and  Bornancin  et  al.  (1980)  have  described  a  microsomal  ATPase 
which  is  stimulated  by  both  HCO3  and  Cr,  and  inhibited  by  thiocyanate.  Im- 
portantly, they  have  shown  that  this  fraction  is  not  contaminated  by  mitochondrial 
anion  ATPase.  They  suggest  that  the  enzyme  is  important  in  Cl~  balance  and  acid/ 
base  regulation  in  fresh  water  since  previous  studies  (DeRenzis,  1975)  had  shown 
that  Cl~  influx  was  correlated  with  base  (presumably  HCO3  )  excretion  and  in- 
hibited by  thiocyanate  (see  above).  It  is  unclear  if  the  enzyme  functions  in  Cr 
transport  in  the  marine  teleosts.  Kormanik  and  Evans  (1979)  have  described  an 
external  HCO3~-sensitive  efflux  of  Cl~  from  O.  beta  in  sea  water,  and  Epstein  et 


FISH  GILL  IONIC  TRANSPORT  125 

al.  (1973)  did  find  that  injection  of  thiocyanate  inhibited  Cl"  efflux  from  seawater 
eels.  However,  the  fact  that  the  activity  of  the  C1~-HCO3~  activated  ATPase  did 
not  change  upon  acclimation  to  sea  water  (Kerstetter  and  Kirschner,  1974;  Bor- 
nancin  et  al.,  1980),  despite  a  significant  difference  in  the  rate  of  Cl~  transport 
across  the  freshwater  vs  seawater  gill  (Evans,  1979)  suggests  that  its  major  role 
may  be  in  the  freshwater  environment.  Indeed,  the  Silva  model  for  NaCl  extrusion 
by  marine  teleosts  (Silva  et  a/.,  1977)  suggests  that  Cl"  exits  the  Cl  cell  down 
electrochemical  gradients  across  the  apical  surface  of  the  "chloride  cell,"  rather 
than  via  Cl  /HCO3~  exchange.  The  latter  cannot  be  ruled  out  at  present  however. 
It  is  clear  that  a  more  detailed  investigation  of  the  role  of  C1~-HCO3~  activated 
ATPase  in  fish  ion  regulation  is  needed. 

THE  ELASMOBRANCHS 

Interest  in  the  rectal  gland  has  nearly  stifled  investigation  of  the  elasmobranch 
branchial  epithelium.  However,  the  ability  of  some  species  to  tolerate  sea  water 
for  prolonged  periods  after  removal  of  the  rectal  gland,  and  the  recent  finding  that 
Na+/NH4+  and  Na+/H+  ionic  exchanges  are  resident  in  the  elasmobranch  bran- 
chial epithelium  (Evans,  et  al.,  1979;  Evans,  1982b)  suggest  that  the  gills  may  play 
some  role  in  salt  extrusion  (see  Evans,  1979  for  a  more  complete  discussion  of  the 
role  of  the  rectal  gland  vs  branchial  epithelium). 

To  a  considerable  extent  the  paucity  of  data  on  the  mechanisms  of  ionic  trans- 
port across  the  elasmobranch  gill  is  secondary  to  their  relatively  large  size,  dis- 
position, and  characteristic  extremely  low  ionic  fluxes  (Evans,  1979).  Nevertheless 
the  few  published  measurements  of  the  TEP  indicate  that  both  Na  and  Cl  are 
maintained  out  of  electrochemical  equilibrium  (Evans,  1980).  We  have  recently 
found  that  prenatal  "pups"  of  the  spiny  dogfish  (Squalus  acanthias}  are  plentiful 
and  easy  to  handle  and  display  the  hallmarks  of  adult  elasmobranch  osmoregulation 
(Kormanik  and  Evans,  1978;  Evans  and  Mansberger,  1979;  Evans  and  Oikari, 
1980).  Importantly  we  have  found  that  the  head  can  be  easily  perfused  and  that 
it  maintains  hemodynamic  stability  for  2-3  hours  (Evans  and  Claiborne,  1982). 
It  is  hoped  that  this  preparation  will  allow  a  more  careful  dissection  of  any  salt 
transport  mechanisms  which  may  reside  in  the  elasmobranch  branchial  epithelium. 

CONCLUSIONS 

It  should  be  obvious  from  this  rather  cursory  review  that  substantial  strides 
have  been  made  in  the  elucidation  of  the  transport  parameters  of  the  fish  gill,  due 
in  no  small  part  to  the  use  of  various  "pieces"  of  the  whole  animal.  To  a  considerable 
extent  the  techniques  have  been  complementary  with  data  from  one  system  cor- 
roborating, but  expanding,  data  from  another  system.  However,  each  of  the  ap- 
proaches has  its  advantages  and  disadvantages,  which  must  be  appreciated  and 
accounted  for.  It  is  also  obvious  that,  per  usual,  the  number  of  species  of  fishes 
which  have  been  examined  is  vanishingly  small,  and  no  single  species  has  been 
examined  utilizing  all  of  the  techniques  described  in  this  review.  It  is  therefore 
appropriate  to  suggest  that  more  species  should  be  examined  and  that  more  in- 
vestigators should  use  a  variety  of  techniques,  rather  than  a  single  method  of 
approach. 

ACKNOWLEDGMENTS 

Our  work  has  been  supported  by  various  grants  from  the  National  Science 
Foundation,  most  recently  PCM  81-04046  to  DHE  and  PCM  80-21971  to  CHM 
as  well  as  NSF  and  NIH  grants  to  the  Mt.  Desert  Island  Biological  Laboratory. 


1  26  DAVID  H.  EVANS  ET  AL. 

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THE  ANATOMY  AND  FINE  STRUCTURE  OF  THE  EYE  IN  FISH.  VI 
CILIARY  TYPE  TISSUE  IN  NINE  SPECIES  OF  TELEOSTS 

D.  EUGENE  COPELAND 
Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

ABSTRACT 

The  eyes  of  teleost  fishes  do  not  have  ciliary  bodies.  Therefore  there  is  no  ciliary 
epithelium  per  se,  the  tissue  normally  assumed  to  secrete  aqueous  humor.  When 
examined  at  the  electron  microscope  level  a  layer  of  nonpigmented  cells  on  the 
back  of  the  fish  iris  shows  many  similarities  to  the  ciliary  epithelium  of  mammals. 
The  tissue  of  fish  iris  has  strategically  located  zonulae  occludents  similar  to  those 
forming  the  blood-aqueous  barrier  in  mammals.  There  is  a  marked  lateral  inter- 
digitation  of  cells  as  seen  in  mammalian  ciliary  tissue  and  as  seen  in  the  specific 
salt  absorbing  cells  found  in  the  gills  of  brackish  water  adapted  crabs.  The  teleost 
tissue  also  has  numerous  intercellular  spaces  (ciliary  channels?)  distributed  in  the 
same  fashion  as  in  mammalian  ciliary  epithelium.  Although  there  is  no  morpho- 
logical evidence  for  the  secretion  of  aqueous  humor,  there  is  indirect  evidence  that 
the  nonpigmented  cells  absorb  salt  to  produce  the  hypotonic  aqueous  humor  that 
is  unique  to  teleosts. 

INTRODUCTION 

The  morphology  of  the  cells  or  tissue  which  secretes  aqueous  humor  in  the  cold 
blooded  vertebrates  has  received  comparatively  little  attention.  Fish  have  been 
almost  completely  neglected.  They  present  an  interesting  problem  in  that  their  eyes 
completely  lack  the  ciliary  muscle  and  associated  epithelium.  Instead,  the  lens  is 
suspended  by  a  membrane  dorsally  and  anchored  ventrally  by  a  hillock  of  muscle 
(campanula  of  Halleri).  The  muscle  retracts  the  lens  to  accomplish  accommodation. 

Zadunaisky  (1972,  1973)  has  studied  the  electrolyte  content  of  the  aqueous 
humor  in  several  fishes  and  has  also  made  preliminary  observations  on  the  possible 
site  of  secretory  origin.  The  epithelium  on  the  posterior  surface  of  the  iris  proves 
to  be  the  likely  source  of  the  primary  aqueous  humor. 

There  is  universal  agreement  that  in  the  higher  vertebrates  the  ciliary  body  in 
some  manner  secretes  the  aqueous  humor.  The  secreted  fluid  then  passes  through 
the  pupil  to  the  anterior  chamber  and  exits  via  the  trabecular  meshwork  and  the 
canal  of  Schlemm.  Evidence  indicates  that  the  secretion  is  accomplished  by  active 
transport. 

The  fine  structure  of  the  ciliary  body  and  its  epithelium  in  mammals  has 
been  thoroughly  investigated.  To  name  a  few  investigators:  Pappas  and  Smelser 
(1961):  Pappas  and  Tennyson  (1962);  Tormey  (1963,  1964);  Kaye  and  Pappas 
(1965);  Bairati  and  Orzalesi  (1966);  Smith  (1971);  Raviola  (1971,  1974);  Shabo 
and  Maxwell  (1972,  1973);  Uusitalo  et  al.  (1973);  Okisaka  (1976a,  b);  Hara  et 
al.  (1977). 

For  reviews  of  the  comparative  composition  of  aqueous  humor  and  the  release 
of  aqueous  humor  see  Cole  (1974),  and  Tripathi  (1974),  respectively. 

Received  14  August  1981;  accepted  12  April  1982. 
Abbreviations:  NPL,  nonpigmented  layer;  PL,  pigmented  layer. 

131 


132  D.  EUGENE  COPELAND 

The  following  report  is  an  expansion  of  Zadunaisky's  initial  studies  and  utilizes 
a  wider  range  of  species  and  different  electron  microscopy  techniques.  It  also  is  a 
continuation  of  my  own  investigations  of  the  eyes  of  fish  (Copeland,  1974a,b,  1976, 
1980;  Copeland  and  Fitzjarrell,  1975;  Copeland  and  Brown,  1976). 

MATERIALS  AND  METHODS 

The  fine  structure  of  the  tissues  on  the  back  surface  of  the  iris  was  investigated 
in  a  wide  variety  of  available  fishes.  The  following  nine  species  were  studied  in 
detail:  blue  gill,  Lepomis  macrochirus  Rafinesque;  eel,  Anguilla  rostrata  (Lesueur); 
mummichog,  Fundulus  heteroclitus  (Linnaeus);  goldfish,  Carassius  auratus  (Lin- 
naeus); scup,  Stenotomous  chrysops  (Linnaeus);  sea  horse,  Hippocampus  erectus 
Perry;  three  spined  stickelback,  Gasterosteus  aculeatus  Linnaeus;  rainbow  smelt, 
Osmerus  mordax  (Mitchill);  rainbow  trout,  Sal  mo  gairdneri  Richardson. 

The  fish  were  narcotized  with  Finquel  (Ayerst  brand  of  tricaine  methane  sur- 
fonate).  If  the  eye  was  difficult  to  enucleate,  a  window  was  cut  in  the  cornea  and 
fixative  introduced  by  blunt  hypodermic  needle  to  the  pupil  in  a  manner  to  gently 
flush  the  back  of  the  iris.  The  eye  was  then  dissected  out  of  the  socket  and  a 
circumferential  cut  made  to  free  the  cornea  plus  part  of  the  sclera  and  retina,  which 
was  then  immersed  in  fixative. 

If  the  eye  was  large  and  easily  removed,  the  circumferential  cut  was  made 
immediately  and  the  front  part  of  the  eye  immersed  then  in  the  fixative. 

The  fixative  was  3%  glutaraldehyde  together  with  1.5%  polyoxymethylene 
(paraformaldehyde)  plus  3%  sucrose  in  0. 1  M  cacodylate  buffer  adjusted  to  pH 
7.4.  Fixation  was  initiated  at  room  temperature  but  as  soon  as  the  dissections  were 
completed  the  vials  were  placed  in  a  refrigerator  for  six  hours.  Final  trimming  was 
done  in  cold  0.1  M  buffer  and  the  tissues  left  in  cold  buffer  several  hours  or  over- 
night. Post  fixation  was  done  with  cold  1%  osmium  tetroxide  in  0.1  M  cacodylate 
for  45  minutes.  The  vials  were  then  brought  to  room  temperature  and  after  several 
buffer  rinses  the  tissues  were  stained  en  bloc  with  2%  uranyl  acetate  in  30%  acetone. 
Dehydration  was  completed  in  acetone  (Baker's  Anhydrous  6- A 137)  and  embed- 
ment done  in  Epon  812.  Sections  were  stained  with  lead  citrate. 

One  of  the  fixative  variations  was  the  use  of  tannic  acid  in  the  first  buffer  rinse 
following  the  primary  fixation  in  an  effort  to  enhance  the  staining  of  the  tissues 
(Simionescu  and  Simionescu,  1976).  Results  were  poor  (probably  inadequate 
penetration)  except  for  one  fortuitous  and  unexpected  result  (see  text  and  Figs.  5 
and  10). 

RESULTS 

The  fine  structure  of  the  tissue  covering  the  back  of  the  iris  in  teleost  fishes  was 
examined.  The  tissue  is  in  the  form  of  a  single  cell  layered  nonpigmented  epithelium 
(NPL)  backed  by  a  layer  of  pigmented  cells  (PL).  The  two  layers  extend  from  the 
retina  to  the  edge  of  the  pupil  and  in  turn  are  backed  by  a  layer  of  connective 
tissue  containing  blood  vessels  (Fig.  1). 

The  most  noticeable  and  consistent  characteristic  of  the  NPL  is  the  baso-lateral 
interdigitations  of  the  cells.  (Note:  During  ontogeny  the  NPL  is  folded  inward  to 
cover  the  PL.  Thus,  the  basal  surface  of  the  NPL  becomes  the  free  surface  in  the 
adult  eye).  The  flattened,  leaf-like  extensions  communicate  to  the  surface  of  the 
epithelium  and  reach  to  varying  depths  within  a  neighboring  cell.  The  cells  mutually 
interdigitate  on  a  one-to-one  basis.  The  occasional  occurrence  of  neighboring  light 
and  dark  cells  demonstrates  this  clearly  (Fig.  4). 


CILIARY  TYPE  TISSUE  IN  FISH 


133 


FIGURE  1.  Stickelback.  Low  power  of  both  the  NPL  and  PL.  The  cell  in  the  NPL  is  covered  by 
an  inner  limiting  membrane  (arrow)  and  shows  numerous  interdigitating  projections  from  a  neighboring 
cell.  The  PL  cell  has  a  basal  lamina  (double  arrow)  and  is  subtended  by  a  blood  vessel  (BV).  Scale:  1.0 
micron. 


The  inner,  vitreal  NPL  bears  an  interesting  relationship  to  the  subtending  PL. 
Peripherally  (i.e.,  toward  the  retina)  the  NPL  is  devoid  of  melanin  granules.  How- 
ever, as  the  pupil  is  approached  there  is  an  increasing  number  of  melanin  granules 
to  be  found  in  the  NPL  until  at  the  pupil  the  two  layers  are  hard  to  distinguish. 


134 


D.  EUGENE  COPELAND 


- 

i 


FIGURE  2.    Smelt.  Showing  exceptionally  large  melanin  granules  in  the  NPL.  Scale:  1.0  micron. 

In  some  instances  the  granules  found  in  the  NPL  enlarge  enormously  (Fig.  2)  and 
in  still  others  they  fragment  (Fig.  3). 

A  much  less  constant  characteristic  is  the  nature  of  the  free  surface  of  the  NPL. 
Most  of  the  species  examined  exhibit  a  relatively  smooth  surface.  However,  goldfish 


FIGURE  3.  Goldfish.  Several  large  intercellular  spaces  (S)  are  seen  between  the  NPL  and  PL 
(ciliary  channels'?).  Multivesiculate  body  (arrow).  Note  fragmentation  of  some  of  the  melanin  granules 
in  the  NPL.  Scale:  1.0  micron. 


CILIARY  TYPE  TISSUE  IN  FISH 


135 


FIGURE  4.     Mummichog.  Low  power  view  of  a  dark  cell  with  contrasting  interdigitations  from 
neighboring  light  cell(s)  of  the  NPL.  Note  the  one-to-one  relationship  of  the  interdigitations.  Scale:  1.0 


mcron. 


(Fig.  3),  blue  gill,  and  trout  have  highly  irregular,  branching  folds  projecting  from 
the  surfaces. 

Another  characteristic  showing  a  degree  of  variation  is  found  in  the  inner  lim- 
iting membrane  on  the  surface  of  the  epithelium.  In  most  fish  it  is  well  developed 
and  strongly  adherent  (goldfish,  Fig.  5  and  eel,  Fig.  6).  In  a  few  it  is  more  fragile 
and  easily  lost  during  the  preparative  procedures  (sea  horse,  Fig.  7).  Due  to  the 
happenstance  of  embryology  of  the  eye,  mentioned  above,  the  membrane  indeed 
is  a  basal  lamina. 

The  mitochondria  of  all  the  cells  are  randomly  distributed  and  showed  no  pref- 
erential orientation  to  the  interdigitated  projections.  However,  they  are  included 
sometimes  within  the  more  blunt  ones  (Fig.  1). 

Well-developed  Golgi  apparati  and  associated  membranous  structures  are  lo- 
cated in  the  scleral  end  of  the  NPL.  Secretory  granules  may  be  seen  in  the  same 
region  (Fig.  8). 


136 


D.  EUGENE  COPELAND 


FIGURE  5.  Goldfish.  Surface  of  the  NPL.  The  inner  limiting  membrane  (ILM)  is  well  developed 
and  adherent.  Penetration  of  tannic  acid  mordant  delineates  the  intercellular  spaces  of  the  interdigitations 
and  the  granular  material  within  the  spaces.  Note  there  are  no  cellular  junctions.  Scale:  0.1  micron. 

Although  not  preserved  in  all  preparations,  coated  vesicles  are  seen  frequently 
(Fig.  9).  Usually,  they  are  found  associated  with  the  free  surfaces  of  the  cells. 

The  NPL  has  both  rough  endoplasmic  reticulum  (usually  in  the  Golgi  area) 
and  smooth  endoplasmic  reticulum  throughout  the  cell. 


FIGURE  6.  £W.  Inner  limiting  membrane  well  developed.  Intercellular  spaces  are  seen  (S).  A 
macula  type  junction  or  desmosome  (arrow)  common  to  the  cell  body  plasma  membranes  (not  to  the 
interdigitations)  is  seen  adjacent  to  one  of  the  spaces.  Scale:  0.1  micron. 


CILIARY  TYPE  TISSUE  IN  FISH 


137 


FIGURE  7.  Sea  Horse.  A  weakly  organized  inner  limiting  membrane  (arrow)  rests  on  the  inter- 
digitations  and  a  few  fibers  common  to  the  vitreous  humor  are  above  the  membrane.  A  high  concentration 
of  filaments  (F)  is  present  within  the  NPL  cell.  A  well-developed  intercellular  space  (asterisk)  is  seen 
near  the  surface.  Scale:  1.0  micron. 


Microtubules  are  seen  occasionally  in  the  cell  surface  areas,  but  much  more 
predominant  are  clusters  of  small  fibrils  (Fig.  10).  In  one  fish,  sea  horse,  they 
occupy  a  good  share  of  the  cell  cytoplasm  (Fig.  7).  They  are  of  the  order  of 
intermediate  or  10  nm  filaments.  That  is,  they  are  "intermediate"  to  microtubules 
at  24  nm  and  microfilaments  at  5-7  nm. 

The  space  between  the  interdigitating  plasma  membranes  of  the  adjoining  cells 
of  the  NPL  is  open  to  the  free  surface.  Due  to  a  fortuitous  usage  of  tannic  acid 
technique,  in  one  instance  the  plasma  membranes  are  not  only  selectively  stained 
but  paniculate  material  is  seen  in  the  intercellular  space  (Figs.  5  and  10).  The 
same  type  of  particles  seen  beneath  the  inner  limiting  membrane  is  found  also 
between  the  cells  (Fig.  5).  Desmosomes  are  found  at  random  intervals  between  the 
plasma  membranes  of  the  cell  bodies  proper,  but  are  seen  rarely  between  the  mem- 
branes of  the  complementary  interdigitations. 


138 


D.  EUGENE  COPELAND 


• 


O 


—L— *r     *L* 


FIGURE  8.  Scup.  Typical  Golgi  apparatus  within  the  NPL  and  adjacent  to  the  PL  interface  (IN). 
Expanded  endoplasmic  reticulum  (E)  with  granular  material.  Secretory  granule  (asterisk).  Ciliary  chan- 
nel (C).  Scale:  1.0  micron. 


The  size  and  number  of  intercellular  spaces  in  the  NPL  varies  from  species  to 
species.  They  may  be  almost  nonexistent,  as  in  stickelback  (Fig.  1),  smelt,  scup, 
mummichog;  small,  eel  (Fig.  6)  and  sea  horse  (Fig.  7);  large,  goldfish  (Fig.  3)  and 
blue  gill;  or  very  large,  trout.  In  some  cases,  spaces  are  seen  also  between  the  NPL 
and  PL  (Fig.  8).  The  spaces  frequently  contain  fine  granular  material  and,  at  times, 
membranous,  multivesiculate  material. 

The  PL  shows  little  activity  compared  to  that  seen  in  mammalian  species.  The 
cells  are  filled  with  melanin  granules  and  have  only  a  few  structures  such  as 
mitochondria,  Golgi  apparati,  endoplasmic  reticulum,  etc.  Occasionally,  a  mild 
degree  of  interdigitation  occurs  between  the  end  of  the  cells  facing  the  vascular 
vessels.  There  also  are  occasional  intercellular  spaces  filled  with  granular  material 
similar  to  that  seen  in  the  spaces  of  the  NPL. 

DISCUSSION 

My  observations  on  the  fine  structure  of  the  goldfish  NPL  are  not  in  complete 
agreement  with  Zadunaisky's  description  of  the  same  species.  His  "microvilli"  are 
in  reality  tortuous  folds  or  outpocketings  of  the  cell  surface.  Also  he  did  not  note 
the  cellular  interdigitations  to  be  found  in  the  NPL  of  the  fish  that  he  describes 
(goldfish).  The  interdigitations  may  not  be  as  numerous  or  complex  as  in  other  fish, 
but  they  are  present. 


CILIARY  TYPE  TISSUE  IN  FISH 


139 


The  interdigitation  of  the  neighboring  cells  is  the  most  consistent  feature  com- 
mon to  all  the  fish  studied.  The  dimensions  of  the  interdigitations  vary  somewhat 
from  species  to  species  but  that  is  not  an  artifact  (i.e.,  exactly  the  same  fixative 
procedures  were  used  throughout).  The  differences  may  be  due  to  slight  differences 
in  the  tonicity  and/or  ionic  balance  in  the  respective  aqueous  humors,  factors  not 
known  at  present. 

Also,  noteworthy  is  the  fact  that  a  plicated  or  ruffled  surface  of  the  NPL  is 
found  only  in  three  fresh  water  forms  (goldfish,  blue  gill  and  trout).  The  surfaces 
are  smooth  in  the  six  sea  water  species  (and  in  a  number  of  other  sea  water  fishes 
not  described  here).  Although  suggestive  of  a  true  difference  beween  fresh  water 
and  sea  water  fish,  a  greater  number  of  fresh  water  fish  would  need  to  be  examined 
to  determine  the  validity  of  such  an  indication. 

The  fine  structure  of  the  epithelium  on  the  back  of  the  teleost  iris  bears  a  close 
and  striking  resemblance  to  the  ciliary  epithelium  of  the  mammals. 


FIGURE  9.    Goldfish.  Tip  of  one  of  the  surface  ruffles  showing  coated  vesicles  (arrow).  Scale:  1.0 
micron. 


140 


D.  EUGENE  COPELAND 


IN 


FIGURE  10.  Goldfish.  NPL  at  its  interface  (IN)  with  the  PL.  From  same  tissue  block  as  in  Figure 
5  but  at  a  lower  power.  Note  that  the  penetration  of  tannic  acid  into  the  intercellular  space  is  limited 
by  the  zonula  occludente  (O).  Cross  section  of  intermediate  filaments  (F).  A  few  microtubules  (asterisk). 
Zonula  adherente  (A).  Scale:  0.1  micron. 


Highly  noteworthy  is  the  existence  of  cellular  interdigitations  in  the  NPL  of 
fish.  These  are  of  a  type  and  orientation  similar  to  those  seen  in  mammals  (ref- 
erences listed  in  the  Introduction).  The  interdigitation  in  teleosts  is  on  a  one-to- 
one  basis  as  demonstrated  by  the  fortuitous  association  of  light  and  dark  cells. 
Tormey  (1963,  1964)  and  Kaye  and  Pappas  (1965)  made  the  same  type  of  obser- 
vations in  the  rabbit  ciliary  epithelium. 

Equally  significant,  the  barrier  of  zonulae  occludentes  and  associated  zonulae 
adherentes  found  at  the  apex  of  the  NPL  of  mammals  (Bairati  and  Orzalesi,  1966; 
Shabo  and  Maxwell,  1972;  Uusitalo  et  al.,  1973;  Raviola,  1974;  Okisaka,  1976b) 
is  found  also  in  fish.  Though  none  of  the  usual  tracers  were  used  in  the  present 
investigation,  the  happenstance  of  limited  tannic  acid  penetration  between  plasma 
membranes  validates  this  interpretation. 

The  NPL  shows  all  the  fine  structure  usually  seen  in  secretory  cells.  There  is 
a  plentiful  supply  of  membranous  organelles  such  as  Golgi  apparati,  endoplasmic 
reticulum  (rough  and  smooth),  mitochondria,  and  granules  filled  with  particulate 
material. 

The  PL  is  packed  with  the  melanin  granules  and  shows  almost  none  of  the 
morphology  usually  associated  with  metabolic  or  secretory  activity.  The  basal-lat- 
eral surfaces  of  the  cells  show  a  mild  degree  of  interdigitation  but  in  no  way 
approach  the  complexity  seen,  for  example,  in  the  monkey  (Okisaka,  1976a). 

One  of  the  prime  "road  blocks"  found  in  the  current  literature  is  the  commonly 
held  belief  that  the  zonulae  occludentes  junctions  in  the  NPL  represent  an  inviolate 


CILIARY  TYPE  TISSUE  IN  FISH  141 

blood-aqueous  barrier.  The  work  of  Raviola  (1974)  gives  most  excellent  support 
to  this  idea.  Nevertheless,  it  should  be  kept  in  mind  that  living  cells  are  dynamic 
systems  and  they  could  well  eliminate  and  reform  junctions  as  they  do  other  or- 
ganelles.  It  is  of  puzzling  significance  that  the  intercellular  spaces  between  the 
NPL  and  PL  (and  frequently  within  the  PL)  have  the  same  appearing  content 
(multivesiculate  or  granular)  as  the  intercellular  spaces  in  the  NPL.  The  finely 
granular  material  is  seen  consistently  enough  to  suggest  that  it  is  a  normally  oc- 
curring material.  However,  the  multivesiculate  type  clusters,  also  sometimes  seen 
in  the  spaces,  occur  randomly  enough  that  they  could  be  artifacts. 

If,  as  repeatedly  stated  in  the  literature,  there  is  indeed  an  inviolate  blood- 
aqueous  barrier  in  the  distal  borders  of  the  NPL  by  reason  of  zonulae  occuludentes, 
then  attention  must  be  turned  to  the  basal  interdigitations  where  only  local,  maculae 
type  junctions  occur  infrequently.  Here  interpretation  of  function,  though  indirect, 
can  be  made  more  plausible  as  explained  below. 

Zadunaisky  (1972)  has  shown  that  the  aqueous  humor  in  two  teleost  fishes 
(goldfish  and  the  marine  sargus)  is  hypotonic  to  the  blood  plasma,  contrary  to  the 
situation  in  mammals  and  amphibia.  His  experimental  physiological  procedures 
indicated  that  sodium  and  potassium  are  preferentially  absorbed  to  effect  the  low- 
ered tonicity.  Later,  at  the  fine  structure  level  he  found  a  histochemical  localization 
of  ATPase  on  the  free  surface  of  the  NPL  (Zadunaisky,  1973).  He  interprets  this 
as  a  possible  site  for  the  metabolic  pump  that  could  account  for  the  absorption  of 
electrolytes  during  the  formation  of  the  hypotonic  aqueous  of  the  fish  eye.  It  is  of 
related  significance  that  Kaye  and  Pappas  (1965)  found  ATPase  on  the  free  surfaces 
and  interdigitations  of  the  equivalent  tissue  in  the  rabbit.  They,  however,  interpret 
the  presence  of  the  enzyme  as  facilitating  the  secretion  of  electrolytes  in  the  for- 
mation of  the  hypertonic  aqueous  of  the  rabbit  eye. 

There  already  exists  an  excellent  example  ol  a  one-to-one  interdigitation  in  a 
tissue  whose  function  is  specifically  osmoregulatory.  The  marine  blue  crab,  Cal- 
linectes  sapidus,  invades  very  dilute  marsh  areas  in  the  warmer  months  in  search 
of  food  and  are  found  in  waters  with  as  low  as  0.5%o  total  salinity.  Salt  is  then 
absorbed  through  an  epithelium  that  lines  a  part  of  the  vascular  space  of  the  gills. 
Physiological  proof  of  salt  absorption  by  crab  gills  was  provided  by  Nagel  (1934) 
and  Koch  et  al  (1954).  The  fine  structure  of  the  single  cell  layered  epithelium 
has  been  studied  by  Copeland  and  Fitzjarrell  (1968).  The  cells  laterally  interdigi- 
tate  quite  deeply  on  a  one-to-one  basis  (see  Fig.  7,  page  8,  Copeland  and  Fitzjarrell, 
1968). 

The  striking  similarity  between  the  crab  gill  tissue,  which  is  specifically  devoted 
to  salt  transport,  and  the  NPL  of  fish  and  mammals  is  remarkable.  This  morpho- 
logical coincidence  plus  the  physiological  determinations  made  by  Zadunaisky 
(1963)  suggest  that  the  NPL  has  an  osmoregulatory  function  related  to  the  ultimate 
producton  of  aqueous  humor. 

Thus,  at  the  morphological  level,  two  functions  can  be  suggested  for  the  com- 
bined NPL  and  PL  of  the  fish  iris.  One,  the  presence  of  similar  intercellular  granular 
material  in  both  the  layers  suggest  a  secretion  of  aqueous  humor  precursor  by  way 
of  the  spaces  between  the  cell  bodies.  Two,  the  plicating  interdigitations  of  the 
NPL  may  refine  the  aqueous  humor  by  means  of  absorbing  specific  electrolytes. 


ACKNOWLEDGMENT 
This  work  was  supported  by  HEW-NEI  grant  number  EY-02647. 


142  D.  EUGENE  COPELAND 

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FUNCTION  OF  CHEMORECEPTOR  ORGANS  IN  SPATIAL 

ORIENTATION  OF  THE  LOBSTER,  HOMARUS  AMERICANUS: 

DIFFERENCES  AND  OVERLAP 

DANA  V.  DEVINE  AND  JELLE  ATEMA 

Boston  University  Marine  Program,  Marine  Biological  Laboratory, 
Woods  Hole,  MA  02543 

ABSTRACT 

Three  of  the  lobster's  main  chemoreceptor  organs,  the  lateral  and  medial  an- 
tennules  (representing  smell)  and  the  dactylus-propodus  segments  of  the  walking 
legs  (representing  taste),  are  physiologically  quite  similar.  We  examined  their  role 
in  spatial  orientation  in  a  food-odor  stimulus  field. 

Control  animals  almost  always  oriented  correctly  and  immediately  to  an  odor 
plume.  Lobsters  with  unilateral  ablations  of  lateral  antennules  lost  this  ability,  but 
did  not  show  preferential  turning  toward  the  intact  side.  Unilateral  medial  anten- 
nule  ablation  did  not  affect  orientation.  Removal  of  all  aesthetasc  hairs  from  one 
lateral  antennule  caused  loss  of  orientation  ability  less  severe  than  unilateral  abla- 
tion of  the  entire  lateral  antennule.  Lobsters  with  unilaterally  ablated  lateral  an- 
tennules and  blocked  walking  leg  receptors  turned  preferentially  toward  the  side 
of  the  intact  antennule. 

Thus,  it  appears  that  intact  lobsters  orient  in  odor  space  by  tropotaxis  principally 
using  aesthetasc  receptor  input.  The  first  two  pairs  of  walking  legs  and  non-aes- 
thetasc  receptors  on  the  lateral  antennule  have  additional  roles  in  spatial  chemical 
orientation.  The  medial  flagellum  does  not  contribute  to  orientation.  Since  loss  of 
appendages  is  relatively  common  in  lobsters,  this  partial  overlap  of  organ  function 
may  serve  the  animal  well  in  nature. 

INTRODUCTION 

In  the  American  lobster,  searching  for  food  may  be  elicited  and  maintained  by 
chemical  cues  alone.  In  order  to  search  efficiently  the  lobster  must  be  able  to 
identify  a  chemical  cue  and  extract  directional  information  from  a  chemical  stim- 
ulus field.  Several  bilateral  chemoreceptor  organs  located  on  different  appendages 
must  be  considered  as  possible  mediators  of  distance  orientation. 

The  biramous  antennules  are  usually  considered  the  distance  chemoreceptors 
in  decapod  crustaceans  (Maynard  and  Dingle,  1963;  Hazlett,  1971).  In  particular, 
the  aesthetasc  hairs  of  their  lateral  flagellum  are  implicated  as  being  chemorecep- 
tors by  morphological  (Laverack,  1964;  Laverack  and  Ardill,  1965;  Ghiradella  et 
al.,  1968),  electrophysiological  (Ache,  1972;  Shepheard,  1974),  and  behavioral 
studies  (McLeese,  1970,  1973,  1974;  Snow,  1973;  Reeder  and  Ache,  1980).  Walk- 
ing leg  and  maxilliped  chemoreceptors  have  been  described  respectively  as  "outer" 
and  "inner"  contact  chemoreceptors  (Luther,  1 930).  On  the  walking  legs  the  regions 
of  greatest  receptor  density  and  specialization  are  the  dactylus  and  propodus  (Derby 
and  Atema,  1982a);  the  walking  leg  chemoreceptors  are  often  incorrectly  called 

Received  1  February  1982;  accepted  21  May  1982. 

144 


LOBSTER  CHEMICAL  ORIENTATION  145 

dactyl  receptors.  Specific  chemoreceptor  sensilla  on  dactylus  and  propodus  have 
been  identified  in  crayfish  (Hatt  and  Bauer,  1980),  and  in  the  lobsters  H.  gammarus 
(Shelton  and  Laverack,  1968,  1970)  and  H.  americanus  (Derby,  1982).  Roles  of 
these  different  chemoreceptor  organs  in  feeding  behavior  of  H.  americanus  were 
described  by  Derby  and  Atema  ( 1 982b).  The  external  appearance  of  chemoreceptor 
sensilla  may  have  been  shaped  by  their  micro-environments;  for  example,  anten- 
nular  chemoreceptors  remain  in  the  water  column,  while  leg  chemoreceptors  are 
subjected  to  abrasion  when  the  lobster  is  walking  or  probing  in  the  substrate 
(Atema,  1980).  Despite  morphological  differences  of  sensilla,  primary  receptor  cells 
of  antennules  and  walking  legs  may  be  quite  similar  in  response  spectrum  and 
threshold.  Although  earlier  studies  found  leg  chemoreceptors  to  have  higher  thresh- 
olds than  antennular  receptors  (Case  and  Gwilliam,  1961;  Ache,  1972;  Shepheard, 
1974;  Fuzessery  and  Childress,  1975;  Fuzessery  et  al.,  1978),  recent  work  has 
shown  that  both  leg  (Derby  and  Atema,  1982a)  and  antennular  receptors  (Thomp- 
son and  Ache,  1980)  can  have  thresholds  lower  than  previously  known.  Based  on 
their  physiology  both  antennules  and  legs  could  be  efficient  distance  chemorecep- 
tors. Studies  which  correlate  this  physiological  and  morphological  information  with 
behavioral  function  are  scarce,  and  the  labels  "contact"  and  "distance"  chemo- 
receptor are  based  on  casual  observation  only.  However,  based  on  neuroanatomical 
and  behavioral  criteria,  the  crustacean  antennules  can  be  called  smell  organs  and 
the  legs  and  maxillipeds  taste  organs  in  analogy  with  vertebrates  and  in  homology 
with  insects  (Atema,  1980). 

An  increase  in  the  rate  of  antennule  flicking,  i.e.,  the  periodic  depression  of  the 
lateral  flagellum  of  the  antennule,  is  generally  one  of  the  first  observable  changes 
in  behavior  after  chemical  stimulation.  This  behavior  has  been  used  to  determine 
chemical  detection  thresholds  (Pearson  and  Olla,  1977).  Thresholds  are  effectively 
lowered  by  flicking  (Schmitt  and  Ache,  1979).  Flicking — functionally  similar  to 
sniffing  by  terrestrial  vertebrates — may  well  compensate  for  the  haphazard  spatial 
and  temporal  character  of  a  chemical  stimulus  field.  This  and  both  physiological 
(Fuzessery,  1978)  and  behavioral  evidence  (McLeese,  1973;  Reeder  and  Ache, 
1980)  strongly  argue  for  the  importance  of  the  lateral  flagellum  in  spatial  orien- 
tation. After  bilateral  ablation  of  the  aesthetasc-bearing  flagella,  spiny  lobsters  did 
not  search  in  response  to  food  odor,  whereas  ablation  of  the  medial  flagella  did  not 
interfere  with  searching  behavior  (Reeder  and  Ache,  1980).  This  animal  uses  both 
tropotactic  and  klinotactic  components  in  orientation  to  food  odors. 

This  study  shows  that  H.  americanus  appears  to  orient  to  odors  principally  by 
means  of  the  aesthetasc  receptor  input,  that  other  chemoreceptors  on  the  lateral 
flagellum  of  the  antennules  may  contribute  somewhat  to  orientation,  and  that  the 
walking  leg  chemoreceptors  function  in  orientation  when  aesthetasc  input  is  im- 
paired. 

MATERIALS  AND  METHODS 
Materials  and  apparatus 

Lobsters  used  in  this  study  (carapace  length  60-82  mm)  were  captured  by  local 
fishermen  in  the  waters  off  Woods  Hole,  Massachusetts.  They  were  maintained  in 
holding  pens  in  running  seawater  for  at  least  two  weeks  prior  to  placement  in 
testing  tanks. 

All  tests  were  done  in  three  identical  675-liter  fiberglass  aquaria  measuring 
1.25  m  long  X  0.9  m  wide  X  0.6  m  deep  with  glass  fronts.  Each  tank  was  outfitted 
with  a  biologically  conditioned  sub-gravel  filter  and  filled  with  either  natural  or 


146  D.  V.  DEVINE  AND  J.  ATEMA 

artificial  seawater.  Illumination  was  provided  by  a  40  W  bulb  suspended  1  m  above 
the  water  surface.  The  light  cycle  approximated  natural  sunrise  and  sunset  for  that 
particular  time  of  year.  The  water  temperature  varied  from  18  to  22°C.  A  single 
animal  was  kept  in  one  tank  for  the  entire  length  of  an  experiment.  Animals  were 
allowed  a  minimum  acclimation  period  of  two  days  before  any  tests  were  run.  The 
lobsters  were  fed  daily  on  a  diet  of  cod  muscle  (Gadus  callarias},  but  never  to 
satiation;  they  were  never  fed  at  the  odor  source  locations  used  in  tests. 

Each  tank  was  fitted  with  a  double  symmetrical  recirculating  seawater  system 
as  described  by  Atema  and  Gagosian  (1973).  The  airlift  water  intakes  were  above 
the  lobster's  shelter,  in  the  rear  center  of  the  tank.  Each  intake  delivered  an  irregular 
flow  of  about  25  ml/sec.  Funnel  interruptions  in  both  sides  of  this  system  allowed 
introduction  of  a  chemical  stimulus  without  appreciable  (2-3%)  concurrent  novel 
mechanical  stimulus.  A  2-4  min  time  delay  further  separated  chemical  stimulus 
arrival  from  possible  contamination  with  a  mechanical  stimulus.  Water  flowed 
down  the  stem  of  the  funnel  to  a  right  angle  glass  elbow  facing  perpendicular  to 
the  side  of  the  tank  and  located  approximately  4  cm  from  the  bottom  of  the  tank. 
This  outflow  was  covered  with  rocks  and  located  inside  the  center  hole  of  a  three- 
hole  cinderblock  to  protect  the  all-glass  system  from  the  test  animals  (Fig.  1). 

The  stimulus  used  was  an  extract  of  homogenized  and  filtered  cod  muscle  at 
a  concentration  of  5  g  wet  weight/1  water.  The  stimulus  side  was  semi-randomly 
switched  between  left  and  right;  however  the  total  number  of  trials  was  divided 
equally  between  the  two  sides.  The  stimulus  was  presented  by  pouring  a  2-ml  dose 
into  the  funnel  over  a  3-sec  interval.  Dye  studies  showed  that  traces  of  the  stimulus 
remained  in  the  funnel  system  for  up  to  30  sec,  and  that  the  dye  pulse  was  visible 
at  the  shelter  between  2  and  4  minutes  after  introduction.  This  variation  in  arrival 
times  was  due  to  variation  in  water  currents  between  tanks;  variation  among  tests 
within  each  tank  was  15-30  sec  (N  =  4).  The  stimulus  pulse  had  been  diluted  by 
a  factor  of  103  to  104  upon  reaching  the  lobster  in  its  shelter  as  measured  by 
colorimetric  analysis  with  methylene  blue  dye.  The  stimulus  front  moving  from 
these  outflows  approached  perpendicular  to  the  length  of  the  lobster  in  the  shelter; 
the  odor  space  appeared  typically  haphazard  with  whirls,  lines,  and  pockets  of 
various  concentrations  of  dye. 

Testing  methods 

Observations  were  made  in  a  darkened  room  with  the  observer  seated  in  front 
of  the  tank.  All  observations  were  made  during  the  day.  Lobsters  are  naturally 
nocturnal.  Light  keeps  them  in  shelter,  but  when  low  enough  it  does  not  prevent 
them  from  searching  when  a  chemical  food  stimulus  is  presented.  All  trials  were 
run  as  described  below. 

Once  the  lobster  was  quiet  and  in  its  shelter,  a  single  stimulus  dose  was  intro- 
duced into  the  tank  via  the  funnel  system.  The  following  three  measurements  on 
orientation  were  made:  latency  to  alert,  initial  direction  choice,  and  search  path. 
The  time  period  from  stimulus  introduction  to  the  lobster's  first  behavioral  response, 
"alert,"  was  recorded.  Among  various  possibilities  we  chose  to  define  "alert"  as 
distinct  waving  and  pointing  of  the  second  (large)  antennae  and  sudden  body  move- 
ments. If  no  alert  response  was  observed  within  8  minutes,  an  animal  was  recorded 
as  giving  "no  response."  Following  alert  and  upon  exit  from  the  shelter,  a  searching 
lobster  made  an  initial  direction  choice,  either  to  the  left  or  right  of  an  imaginary 
line  down  the  center  of  the  tank.  References  to  handedness  always  refer  to  the 
perspective  of  a  lobster  in  its  shelter,  not  to  the  observer.  This  initial  direction 


LOBSTER  CHEMICAL  ORIENTATION 


147 


FIGURE  1.     Diagram  of  test  aquarium:  (1)  chemical  stimulus,  (2)  air  lift,  (3)  brick,  (4)  gravel 
substrate. 


choice  was  scored  as  either  "+"  or  "-  '  with  respect  to  the  stimulus  side  or  "no 
response"  if  searching  was  not  elicited.  A  sketch  was  made  of  the  path  taken  by 
the  test  animal  while  it  searched  for  the  stimulus  source.  A  test  was  considered 
completed  when  either  an  animal  located  the  stimulus  source  or  10  minutes  had 
elapsed  since  the  introduction  of  the  stimulus.  The  search  path  lengths  were 
subsequently  measured  with  a  planimeter  and  converted  back  to  actual  distances 
walked  by  the  test  animal.  Although  preliminary  tests  showed  that  a  lobster's 
performance  was  not  affected  by  tests  as  close  together  as  1.5  hours,  3  to  12  hours 
were  generally  allowed  between  tests.  Two  to  four  tests  were  run  per  animal 
per  day. 

Treatment  groups 

The  experiments  were  organized  into  four  treatment  groups  of  six  lobsters  each. 
In  all  groups,  fifteen  trials  were  run  per  animal  to  establish  a  baseline.  The  same 
number  of  trials  was  run  on  each  animal  after  each  phase  of  treatment.  The  treat- 
ments were:  1 )  The  right  lateral  antennular  flagellum  was  ablated,  then  the  right 
medial  flagellum  was  subsequently  ablated.  2)  The  sequence  of  treatment  1  was 
reversed:  ablation  of  the  right  medial  flagellum  was  followed  by  right  lateral  an- 
tennular ablation.  3)  The  aesthetasc  hairs  of  the  right  lateral  flagellum  were  shaved 
off  with  a  scalpel  while  the  lobster  was  cold-anaesthetized  on  its  back  in  a  tray  of 
ice.  Once  the  experiment  was  completed,  the  shaved  flagella  were  removed  and 
prepared  for  scanning  electron  microscopy.  4)  The  dactylopodite  and  propodite  of 
all  four  pairs  of  walking  legs  as  well  as  the  seizer  claw  were  coated  with  the 
cyanoacrylate  adhesive  KrazyGlue®  which  formed  a  waterproof  acrylic  "glove" 
around  the  leg  when  dry.  The  use  of  cyanoacrylates  does  not  result  in  non-specific 
behavioral  changes  in  H.  americanus  (Derby  and  Atema,  1982b;  C.  Derby,  un- 
published data).  These  animals  underwent  subsequent  ablation  of  the  right  lateral 
antennular  flagellum.  The  right  side  of  all  animals  was  consistently  used  for  ablation 
since  there  is  no  evidence  for  dominance  of  left  or  right  in  processing  chemosensory 
input.  Eighteen-hour  recovery  periods  were  allowed  following  all  treatments  for 
recovery  from  the  effects  of  ablation  and  handling. 

Friedman's  analysis  of  variance  by  ranks  (Xr2)  and  multiple  comparisons  for 
ranked  data  were  performed  using  the  sums  of  each  fifteen-trial  group  (Zar,  1974). 


148 


D.  V.  DEVINE  AND  J.  ATEMA 


For  presentation  purposes,  means  and  standard  errors  were  calculated  for  each 
treatment  group.  Significance  was  accepted  at  the  0.05  level. 

RESULTS 

Normal  lobsters  initially  responded  to  cod  muscle  extract  by  waving  their  an- 
tennae, increasing  the  rate  of  antennule  flicking,  and  changing  body  stance  and 
claw  posture  from  resting  to  walking.  Following  this  alert  response  and  a  short  wait 
in  the  shelter,  the  lobster  left  the  shelter  in  search  of  the  odor  source.  Initial  direction 
choice  was  almost  always  correct  (347  times  out  of  360  tests).  Searching  consisted 
of  walking  a  generally  straight  path  to  the  odor  source  while  doing  much  antenna 
waving,  antennule  flicking,  fanning  the  exopodites  of  the  maxillipeds,  pleopod  beat- 
ing, and  occasional  wiping  of  antennules  by  the  third  maxillipeds.  Within  one  body 
length  of  the  odor  source,  the  animals  often  probed  the  substrate  with  walking  legs 
and  maxillipeds.  Upon  reaching  the  odor  source,  normal  animals  would  attempt 
to  reach  the  outflow  by  inserting  their  first  and  second  walking  legs  into  the  cin- 
derblock  where  the  odor  outflow  was  hidden.  They  often  persisted  in  this  directed 
search  behavior  for  several  minutes. 

None  of  the  treatments  affected  either  the  lobster's  use  of  antennae  and  walking 
legs  in  searching  or  their  probing  with  walking  legs  and  moving  of  maxillipeds. 
Also,  none  of  the  treatments  affected  alert  latency  (Tables  I  and  II).  However,  in 


TABLE  I 
Effects  of  chemorec eptor  appendage  ablations  on  three  behavioral  parameters. 


Alert  latency 
(sec) 
X  ±  SEM 

Correct  direction 
choice 
(15  trials) 

Search 
path  length  (cm) 

Group  1 

a)  Untreated 

106.7  ±  9.5 

14.3  ±  0.3 

159.3  ±  30.0 

b)  Right  lateral  ablation 

102.2  ±  9.8 

7.8  ±  0.3 

250.5  ±  34.3 

c)  Subsequent  right 

medial  ablation 

91.5  ±  14.9 

8.0  ±  0.4 

218.5  ±  33.5 

Statistic  and  significance 

Xr2  -  5.33  NS 

Xr2  =  9.08  P  <  0.02 

Xr2  ==  10.3  P  <  0.01 

Group  2 

a)  Untreated 

153.0  ±  16.0 

14.2  ±  0.3 

105.3  ±  4.7 

b)  Right  medial  ablation 

164.2  ±  17.9 

14.8  ±  0.2 

103.5  ±  3.7 

c)  Subsequent  right 

lateral  ablation 

171.2  ±  21.2 

8.8  ±  0.5 

209.2  ±  14.8 

Statistic  and  significance 

Xr2   =  2.3    NS 

Xr2  =  9.34  P  <  0.01 

Xr2  =  9.33  P  <  0.01 

Group  3 

a)  Untreated 

171.2  ±  14.4 

14.7  ±  0.2 

111.7  ±  10.0 

b)  Right  aesthetascs 

shaved 

195.7  ±  20.0 

9.8  ±  0.8 

236.8  ±  35.5 

Statistic  and  significance 

Xr2   =  2.6    NS 

Xr2  =6       P  <  0.05 

Xr2  =  6        P  <  0.05 

Group  4 

a)  Untreated 

167.3  ±  20.7 

14.7  ±  0.2 

98.0  ±  3.3 

b)  Glue-covered  legs 

172.8  ±  24.0 

14.8  ±  0.2 

110.2  ±  4.9 

c)  Subsequent  right 

lateral  ablation 

180.8  ±  22.2 

8.3  ±  1.0 

271.3  ±  12.9 

Statistic  and  significance 

Xr2   =  5.33  NS 

Xr2  =  9.0    P  <  0.02 

Xr2  =  9.33  P  <  0.01 

NS  =  not  significant. 


LOBSTER  CHEMICAL  ORIENTATION 


149 


TABLE  II 
Behavioral  changes  in  food  odor  orientation  after  chemoreceptor  appendage  ablations. 


Correct 

Change  in 

Change  in 

direction 

search  path                 Circus 

Group 

Ablations 

alert  latency 

choice  (%) 

length  (%)              movement 

la 

None 



94 

—                          no 

Ib 

Lateral  (L) 

no 

|52°U 

+71**h 

no 

Ic 

L  +  M 

no 

53° 

+44** 

no 

2b 

Medial  (M) 

no 

99           * 

+  1 

no 

2c 

M  +  L 

no 

59** 

+101** 

**              no 

3b 

Aesthetasc 

no 

|66*H 

+  108* 

no 

4b 

Legs  Coated  (C) 

no 

99 

+  14 

no 

4c 

C  +  L 

no 

56° 

+  177**^                   yes 

Group  numbers  and  treatments  are  the  same  as  in  Table  I. 

Statistical  significance:  *P  <  0.05,  °P  <  0.02,  **/>  <  0.01  (Mann-Whitney  U-test). 


all  treatment  groups,  both  initial  direction  choice  and  subsequent  search  path  length 
were  significantly  altered  by  ablation  of  the  right  lateral  flagellum  or  by  removal 
of  aesthetasc  hairs  regardless  of  the  presence  (groups  Ib,  3b,  and  4c)  or  absence 
(group  2c)  of  the  medial  flagellum.  In  contrast,  glue-coating  all  walking  legs  (group 
4b)  did  not  change  initial  direction  choice  nor  search  path  length  (Tables  I  and 
II).  and  ablation  of  the  medial  flagellum  before  (group  2b)  or  after  (group  Ic) 
ablation  of  the  lateral  flagellum  had  no  effect  on  any  of  the  measured  behavioral 
parameters. 

SEM  showed  that  the  aesthetasc  hairs  of  animals  in  treatment  group  3  were 
indeed  removed.  Even  in  the  least  effective  shaving  (Fig.  2),  only  the  bases  of  some 
sensilla  remained.  The  significant  decrease  in  the  number  of  correct  initial  direction 
choices  and  the  concomitant  increase  in  search  path  length  caused  by  aesthetasc 
shaving  was  not  as  great  as  the  decrease  caused  by  lateral  flagellum  removal;  the 
difference  is  significant  in  itself  (Table  II). 

In  contrast  to  lobsters  in  other  treatments,  only  leg-coated  lobsters  with  uni- 
lateral ablation  of  the  right  lateral  flagellum  (group  4c)  made  a  significantly  higher 
number  of  initial  direction  choices  to  the  left  (i.e.  intact)  side  (Xr2  =  9.33;  P 
<  0.01)  regardless  of  stimulus  direction.  In  many  of  the  trials  these  lobsters  made 
complete  left-turning  circles  while  searching  (circus  movements,  Table  II).  Besides 
becoming  more  erratic  these  animals  searched  more  slowly,  and  the  increase  in 
length  of  their  search  paths  was  significantly  greater  (P  <  0.0 1 )  than  that  of  lobsters 
with  only  lateral  flagellum  ablations  (Table  II).  Outside  the  experimental  obser- 
vation regime,  during  feeding  such  lobsters  had  difficulties  locating  their  daily  food. 

DISCUSSION 

Since  none  of  the  experimental  manipulations  altered  alert  latency,  and  assum- 
ing that  latency  across  animals  and  experiments  is  correlated  with  detection  thresh- 
old, we  conclude  that  the  lobsters'  threshold  for  odor  detection  and  identification 
at  this  stimulus  concentration  was  not  affected  by  unilateral  antennule  ablations 
and/or  glue-covered  legs.  The  results  of  experiments  1  and  2  demonstrate  the 
importance  of  lateral  flagellar  chemoreceptors  for  the  extraction  of  directional 
information  from  a  chemical  stimulus  field.  This  is  reflected  in  the  dramatic  shift 
in  correct  initial  direction  choice  from  nearly  100%  to  roughly  random  following 


150 


D.  V.  DEVINE  AND  J.  ATEMA 


FIGURE  2.  SEM  of  portion  of  lateral  flagellum  of  antennule.  Top:  (A)  Normal  rows  of  8-12 
aesthetasc  hairs,  2  rows  per  segment,  flanked  by  much  larger  guard  hairs.  Bottom:  (B)  Aesthetasc  and 
guard  hairs  shaved  off  at  base;  in  this  worst  example  several  remaining  aesthetasc  stumps  contain  only 
proximal  segments  of  receptor  dendrites. 


LOBSTER  CHEMICAL  ORIENTATION  1  5  1 

unilateral  ablation  of  the  lateral  flagellum.  The  same  effects  are  seen  when  search 
path  length  is  used  as  a  measurement  of  orientation  efficiency.  Ablation  of  the 
ipsilateral  medial  flagellum  either  prior  to  or  following  lateral  ablation  had  no 
effect  on  searching  behavior,  indicating  that  the  input  from  medial  flagellum  re- 
ceptors was  not  necessary  for  efficient  orientation  to  odors. 

Removal  of  aesthetasc  sensilla  alone  was  sufficient  to  cause  significant  changes 
in  orientation  ability,  but  it  did  not  affect  initial  direction  choice  as  much  as  entire 
lateral  flagellum  ablation  (Table  II).  Therefore,  input  from  other,  unidentified 
lateral  flagellum  chemoreceptors  may  aid  in  orientation.  There  is  both  physiological 
(Fuzessery,  1978)  and  morphological  evidence  (Laverack,  1964;  Derby,  1982;  Glee- 
son,  1982)  for  the  existence  of  non-aesthetasc  receptors.  Since  the  lateral  flagellum 
is  adapted  to  temporal  and  spatial  sampling  of  odor  space  through  a  combination 
of  its  morphology  and  flicking  behavior  (Schmitt  and  Ache,  1979),  one  could  hy- 
pothesize that  the  entire  chemosensory  input  from  this  flicking  appendage  is  useful 
for  spatial  orientation.  Yet,  the  aesthetasc  sensilla  probably  carry  the  bulk  of  the 
information  if  only  by  the  sheer  number  of  their  afferent  neurons,  about  400  per 
sensillum  (Oleszko-Szuts  and  Atema,  1977).  The  behavioral  experiments  reported 
here  support  this  notion  and  extend  the  results  of  antennule  impairments  obtained 
for  spiny  lobsters  (Reeder  and  Ache,  1980)  by  identifying  the  aesthetasc  input  as 
the  main  but  not  the  exclusive  source  of  directional  information.  The  alternate 
explanation  that  the  remaining  aesthetasc  hair  bases  retain  partial  function  cannot 
be  rejected,  but  is  in  our  opinion  less  likely,  based  on  electron  microscopic  obser- 
vation of  receptor  morphology.  The  base  of  the  aesthetasc  hair  is  made  of  thick, 
lamellated  cuticle,  lined  inside  with  supporting  cells.  At  the  level  of  the  transition 
from  the  base  to  the  distal  portion,  inside  the  hair  are  the  ciliary  junctions  of  the 
receptor  cells  (Oleszko-Szuts  and  Atema,  1977).  Thus  all  of  the  receptor  cell  distal 
segments  were  removed  in  the  incomplete  shavings.  Interactions  of  stimulus  and 
receptor  molecule  presumably  occurs  in  the  ciliary  distal  segments. 

If  lobsters  are  using  tropotaxis  to  make  their  initial  direction  choice,  unilaterally 
ablated  animals  would  be  expected  to  show  preferential  turning  toward  the  intact 
side  regardless  of  the  direction  of  the  stimulus  (Fraenkel  and  Gunn,  1961 ),  resulting 
in  circus  movements,  i.e.  turning  circles  in  the  direction  of  the  intact  side  when 
stimulated.  Such  circus  movements  in  chemical  gradients  were  described  for  spiny 
lobsters  with  unilateral  antennule  ablation  (Reeder  and  Ache,  1980)  but  they  were 
not  seen  in  Homarus  americanus  (McLeese,  1973).  In  our  experiments  preferential 
turning  to  the  intact  side  and  circus  movements  (Table  II)  were  only  seen  when 
laterally  ablated  lobsters  also  had  the  propodus  and  dactylus  of  their  walking  legs 
coated  with  glue.  Such  coated  and  ablated  lobsters  also  showed  greater  search  path 
errancy  compared  to  lobsters  with  only  lateral  flagellum  ablations  (Table  II).  These 
results  demonstrate  that  leg  receptor  input  does  play  a  secondary  role  in  spatial 
orientation  in  a  chemical  stimulus  field;  this  role  becomes  apparent  when  antennular 
chemoreception  is  disrupted.  However,  in  otherwise  intact  lobsters,  leg  chemore- 
ceptor  input  was  not  essential  for  efficient  orientation. 

The  possibility  of  chemically  stimulated  rheotaxis  can  not  be  overlooked.  Under 
this  hypothesis,  a  lobster  is  stimulated  by  the  biologically  significant  odor  of  food 
to  search  with  mechanoreceptors  in  the  ever-present  flow  gradients.  In  general,  the 
chemical  senses  are  closely  allied  with  mechanoreceptors,  both  morphologically  and 
functionally. 

In  animals  such  as  lobsters,  which  regularly  lose  appendages  to  predators,  in 
social  interactions,  or  through  molting  disturbance,  overlap  and  redundancy  of 
sensory  input  must  be  very  important.  Our  results  indicate  that  when  the  main 


152  D.  V.  DEVINE  AND  J.  ATEMA 

chemosensory  input  to  spatial  orientation  is  lost,  other  inputs  can  take  over,  at 
least  partially.  This  behavioral  recovery  of  function  was  not  complete  in  the  short 
duration  of  these  experiments  (days),  but  may  well  improve  over  time  (weeks)  as 
suggested  by  studies  on  hermit  crabs  (Hazlett,  1971)  and  lobsters  (Atema 
et  al,  1981). 

ACKNOWLEDGMENTS 

We  thank  Dr.  Charles  Derby  for  his  SEM  assistance  (Fig.  2b),  for  critical 
reading  of  the  manuscript,  and  for  many  stimulating  discussions.  We  thank  Thomas 
Trott  for  his  help  in  various  phases  of  the  research  and  for  review  and  discussion 
of  the  manuscript.  This  study  was  supported  in  part  by  the  Department  of  Biology, 
Boston  University,  and  by  D.O.E.  Grant  #EY76S022546  to  J.A. 

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SOMATOTOPY  IN  THE  REPRESENTATION  OF  THE  PECTORAL  FIN 
AND  FREE  FIN  RAYS  IN  THE  SPINAL  CORD  OF  THE  SEA  ROBIN, 

PRIONOTUS  CAROLINUS 

THOMAS  E.  FINGER 

Marine  Biological  Laboratory,  Woods  Hole,  MA  02543,  and  *  Department  of  Anatomy. 
University  of  Colorado  Medical  School,  Denver,  CO  80262 

ABSTRACT 

Sea  robins  possess  modified  pectoral  fin  rays  which  are  chemosensory  although 
lacking  taste  buds  or  olfactory  receptors.  These  fin  rays  are  innervated  only  by 
spinal  nerves  which  terminate  in  accessory  spinal  lobes,  enlargements  of  the  dorsal 
horn  in  the  rostral  spinal  cord. 

Horseradish  peroxidase  was  used  as  a  neuronal  tracer  to  determine  the  rep- 
resentation of  each  fin  ray  nerve  in  the  spinal  cord.  The  nerve  innervating  each  fin 
ray  terminates  in  a  single  accessory  lobe  with  the  ventral  fin  ray  terminating  in  the 
caudal  accessory  lobe  and  the  dorsal  fin  ray  in  the  rostral  major  accessory  lobe. 
The  pectoral  fin  itself  is  represented  in  a  minor  spinal  enlargement  lying  rostral 
of  the  major  accessory  lobes. 

INTRODUCTION 

Sea  robins  (Prionotus)  and  the  related  European  gurnards  (Triglida]  possess 
modified  pectoral  fins  which  are  capable  of  detecting  low  levels  of  certain  chemicals 
despite  the  absence  of  taste  buds  or  other  specialized  chemosensory  end  organs 
(Whitear,  1971).  In  these  genera,  the  three  most  ventral  fin  rays  are  free  from  the 
rest  of  the  pectoral  fin  (Fig.  1 ).  The  free  fin  rays  are  moved  independently  of  the 
fin  and  are  used  actively  to  explore  the  substrate  (Morrill,  1895;  personal  obser- 
vation). The  fish  will  respond  positively  when  the  free  fin  rays  contact  food  or  food 
extracts  (Bardach  and  Case,  1965). 

Despite  the  fact  that  the  free  fin  rays  are  used  to  locate  food,  the  fin  rays  possess 
no  taste  buds  (or  olfactory  receptors),  and  are  innervated  only  by  spinal  nerves 
(Morrill,  1895).  Whitear  (1971)  confirms  that  no  specialized  chemosensory  end 
organs  occur  on  the  fin  rays  although  numerous  isolated  chemosensory  cells  lie  in 
the  skin.  The  fin  ray  nerves  emerge  from  the  fused  dorsal  root  ganglion  of  the 
second  and  third  spinal  roots  (Herrick,  1907;  also  see  Fig.  2).  The  nerves  innervating 
the  pectoral  fin  proper  also  emerge  from  this  same  ganglionic  mass. 

At  the  level  of  entrance  of  these  nerve  roots  into  the  spinal  cord,  three  major 
paired  accessory  spinal  lobes  occur  on  the  dorsal  aspect  of  the  spinal  cord.  Herrick 
(1907)  describes  these  accessory  lobes  as  enlargements  of  the  dorsal  horn  of  the 
spinal  cord.  In  addition,  smaller  swellings  occur  farther  rostral  in  the  spinal  cord. 
Because  the  numbering  system  used  by  Herrick  does  not  correspond  to  the  patterns 
of  lobulation  observed  in  the  live  specimens  (to  which  Herrick  did  not  have  access), 


Received  16  February  1982;  accepted  29  March  1982. 
*  Address  to  which  reprint  requests  should  be  addressed. 

Abbreviations:  HRP,  horseradish  perioxidase;  HY,  Hanker- Yates  peroxidase  method;  TMB,  tet- 
ramethyl  benzidine. 

154 


SEA  ROBIN  SPINAL  CORD 


155 


FIGURE  1.    Photograph  of  a  sea  robin  showing  the  free  fin  rays  and  pectoral  fin.  D,  dorsal  fin  ray; 
M,  middle  fin  ray;  PF,  pectoral  fin;  V,  ventral  fin  ray.  Approximately  2/3  life  size. 

the  lobes  have  been  renumbered  in  this  work,  according  to  the  scheme  indicated 
in  Figure  2.  The  major  accessory  lobes  are  numbered  1-3,  with  number  1  being 
the  most  caudal.  Accessory  lobe  4  is  a  much  smaller  swelling  located  immediately 
rostral  to  the  major  accessory  lobes.  For  reasons  given  elsewhere  (T.  Finger,  in 
preparation)  and  below,  the  other  swellings  on  the  surface  of  the  rostral  spinal  cord 
should  not  be  considered  homologous  to  the  accessory  lobes  1-4  described  above 
and  are  not  simple  elaborations  of  the  dorsal  horn  of  the  spinal  cord. 


FIGURE  2.  Photograph  of  the  brain  and  spinal  ganglion  of  a  sea  robin.  White  arrowhead  indicates 
the  sulcus  of  the  accessory  lobe  wherein  a  blood  vessel  lies.  1,  2,  3,  4  indicate  the  accessory  lobes  of  the 
spinal  cord.  Cb,  cerebellum;  Fb,  forebrain;  G,  dorsal  root  ganglion  for  the  fin  ray  nerves;  TeO,  optic 
tectum.  Bar  scale  equals  5  mm. 


156  THOMAS  E.  FINGER 

The  pathways  and  nuclei  of  the  central  nervous  system  involved  in  the  spinal 
chemical  sense  have  not  been  studied  with  modern  anatomical  techniques.  This 
first  in  a  series  of  papers  on  the  common  chemical  sense  in  sea  robins  utilizes 
neuroanatomical  tracing  techniques  to  determine  the  representation  of  the  pectoral 
fin  and  free  fin  rays  in  the  rostral  spinal  cord.  The  pectoral  fin  nerves  project  to 
the  minor  accessory  lobe  (number  4)  while  the  free  fin  rays  project  to  the  major 
accessory  lobes  (numbers  1-3). 

MATERIALS  AND  METHODS 

Live  sea  robins  (Prionotus  carolinus}  were  obtained  through  the  collection 
service  at  the  Marine  Biological  Laboratory,  Woods  Hole,  MA.  The  animals  were 
fed  pieces  of  squid  and  maintained  in  holding  tanks  supplied  by  running  water. 

Operations  were  carried  out  on  fish  which  were  anaesthetized  with  tricaine 
methane-sulfonate  (MS-222).  Initially,  the  animals  were  placed  in  seawater  con- 
taining a  1:10,000  dilution  of  the  drug.  When  opercular  movements  were  barely 
perceptible  or  had  ceased,  the  fish  was  transferred  to  an  operation  chamber  which 
held  the  animal  semirigidly  by  means  of  modelling  clay  blocks.  The  fish  was  covered 
with  wet  gauze  and  a  recirculating  pump  supplied  water  to  a  tube  inserted  in  the 
animal's  mouth.  The  water  in  the  operating  chamber  contained  anaesthesia  at  a 
dilution  of  1:20,000-1:40,000  depending  on  the  anticipated  duration  of  the  pro- 
cedure; higher  concentrations  were  necessary  for  the  longer  operations.  Following 
the  surgery,  the  fish  was  placed  in  its  home  tank  and  revived  by  placing  its  mouth 
over  the  inlet  tube  for  incoming  seawater. 

Horseradish  peroxidase  (HRP,  Sigma  Type  VI)  was  used  as  an  anterograde 
or  transganglionic  tracer.  A  30-50%  solution  of  HRP  was  prepared  in  a  1%  solution 
of  a-lysophosphatydal  choline  (lysolecithin).  The  tracer  was  applied  in  one  of  two 
fashions.  For  ganglionic  injections,  HRP  paste  was  applied  to  the  end  of  a  size  00 
insect  pin  which  was  then  inserted  repeatedly  into  the  ganglionic  mass  (Finger, 
1976).  For  applications  to  a  peripheral  nerve,  the  nerve  was  exposed  and  isolated 
on  gelfoam.  A  small  piece  of  gelfoam  soaked  in  HRP  was  then  placed  on  the  cut 
end  of  the  nerve.  Fine  forceps  and  an  insect  pin  were  used  to  divide  the  nerve  into 
numerous  fascicles  which  then  were  threaded  through  the  HRP-gelfoam.  In  some 
cases,  flattened  sheets  of  styrofoam  (from  a  hot-cup)  were  sandwiched  around  the 
nerve-gelfoam  and  the  entire  assembly  glued  (cyano-acrylate  glue;  histo-acryl  or 
Crazy  Glue®)  back  into  place  beneath  the  skin.  The  overlying  skin  was  then  sutured 
and  glued  to  form  a  watertight  covering. 

A  total  of  12  fish  was  used  in  this  study,  but  because  some  fish  had  two  nerves 
labeled,  one  on  each  side,  these  animals  represent  18  experiments.  In  four  cases, 
the  dorsal  roots  were  labeled  by  intraganglionic  injections  of  HRP.  These  cases 
provided  the  clearest  labeling  of  the  primary  afferent  terminals.  The  remaining  14 
experiments  entailed  applications  of  HRP  to  the  peripheral  nerve  as  follows:  ventral 
fin  ray,  three  cases;  middle  fin  ray,  three  cases;  dorsal  fin  ray,  four  cases;  superior 
pectoral  fin  nerve,  two  cases;  inferior  pectoral  fin  nerve,  two  cases. 

Following  survival  times  of  1-2  days  for  ganglionic  injections  (four  cases)  and 
4-10  days  for  peripheral  nerve  injections  (eight  animals,  14  nerves),  the  fish  were 
reanaesthetized  and  perfused  through  the  conus  arteriosus  with  20  ml  of  saline 
followed  by  50-100  ml  of  4%  glutaraldehyde  in  phosphate  buffer  (0.1  M,  pH  7.2). 
The  brain,  rostral  spinal  cord,  and,  in  some  cases,  spinal  ganglia  were  removed 
from  the  animal,  encased  in  gelatin  (Finger,  1976)  and  fixed  an  additional  3-6 
hours.  The  gelatin  blocks  were  washed  in  phosphate  buffer  and  stored  overnight 


SEA  ROBIN  SPINAL  CORD  1  57 


B 


FIGURE  3.  Photomicrographs  and  chartings  of  transverse  sections  through  the  accessory  lobes. 
(A)  Major  accessory  lobe.  Note  the  prominent  outer  parvocellular  layer  surrounding  the  lobe.  (B)  Minor 
accessory  lobe.  The  position  of  two  lobules  is  indicated.  AcLM,  major  accessory  lobe;  AcLmn,  minor 
accesory  lobe;  DH,  dorsal  horn;  Fdl,  dorsolateral  fasciculus;  Flm,  medial  longitudinal  fasciculus;  fV, 
ventral  fasciculus;  Lbl,  lobule;  MP,  segmental  spinal  motor  neuron  pool;  SpN,  spinal  nerve  root. 


in  buffer  containing  10-20%  sucrose.  The  tissue  was  sectioned  at  35  ^m  in  either 
the  horizontal  or  transverse  planes,  on  a  freezing,  sliding  microtome.  The  sections 
were  reacted  for  the  presence  of  peroxidase  by  means  of  a  modified  Hanker-Yates 
(HY)  method  (Bell  et  al,  1981)  or  by  the  tetramethylbenzidine  (TMB)  method 
of  Mesulam  (1978).  In  many  cases  alternate  sections  were  reacted  using  the  two 
different  methods.  The  reaction  product  from  the  TMB  method  was  visualized 
more  easily,  by  darkfield  microscopy,  and  the  TMB  reaction  was  slightly  more 
sensitive.  However,  the  HY  method  produced  a  less  granular  reaction  product 
which  better  revealed  fine  structural  details  of  the  labeled  fibers  and  cells. 

RESULTS 

Pattern  of  peripheral  innervation.  The  three  free  fin  rays  are  each  innervated 
by  a  unique  branch  arising  from  the  fused  ganglion  of  the  second  and  third  spinal 
roots  (Morrill,  1895;  Herrick,  1907).  The  fin  ray  nerves  form  separate  fascicles 
within  1  cm  of  the  ganglion,  somewhat  dorsal  to  the  pectoral  fin  proper. 

Two  other  major  nerves  leave  this  same  ganglionic  mass  to  innervate  the  pectoral 
fin  proper.  A  large  nerve  turns  rostrally  from  the  ganglion  and  enters  the  pectoral 
fin  from  its  superior,  or  anterodorsal,  aspect.  This  nerve  is  termed  the  superior 
pectoral  fin  nerve.  The  smaller  nerve  innervating  the  pectoral  fin,  the  inferior  pec- 
toral fin  nerve,  travels  with  the  fin  ray  nerves  caudal  to  the  pectoral  fin  but  turns 
rostrally  to  innervate  the  inferior  face  of  the  pectoral  fin.  The  fin  ray  nerves  continue 
ventrally  to  reach  the  free  fin  rays.  Immediately  before  entering  the  fin  ray,  a  given 
fin  ray  nerve  splits  into  two  branches,  one  branch  innervating  the  surface  rostral 
to  the  cartilagenous  core  of  the  fin  ray,  and  one  branch  innervating  caudal  to  the 
cartilagenous  core.  No  attempts  were  made  in  the  present  study  to  trace  separate 
connections  of  the  anterior  and  posterior  branches  of  each  fin  ray  nerve. 

Structure  of  the  accessory  lobes.  The  major  spinal  accessory  lobes  (numbers 
1-3)  each  are  divided  in  half  along  their  rostrocaudal  axis  by  an  indentation  along 
the  dorsal  surface.  This  superficial  groove  often  embraces  a  blood  vessel.  No  similar 
division  of  the  minor  accessory  lobe  (lobe  4)  occurs  although  blood  vessels  do  run 
across  the  surface  of  this  structure  as  well. 

All  of  the  accessory  lobes  contain  an  outer  layer  (approx.  40  yum  thick)  of  small 
neurons  and  an  inner  zone  of  neurons  mixed  with  neurophil  (Fig.  3A).  Golgi  prep- 


158 


THOMAS  E.  FINGER 


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FIGURE  4.  Photomicrographs  of  terminals  and  preterminal  arborizations  in  the  spinal  accessory 
lobes  following  intraganglionic  injection  of  HRP.  Hanker-Yates  reaction.  (A)  Numerous  branches  and 
en  passant  swellings  occur  in  the  subparvocellular  layer  (sp).  p,  parvocellular  layer.  (B)  An  apparent 
terminal  among  the  small  neurons  of  the  parvocellular  layer. 


arations  reveal  that  most  dendrites  of  neurons  in  the  accessory  lobes  are  oriented 
radially.  The  detailed  structure  of  the  lobes  will  be  discussed  in  a  later  paper  (T. 
Finger,  in  preparation).  The  minor  accessory  lobe  (lobe  4)  comprises  a  number  of 
lobules,  each  of  which  is  surrounded  by  a  parvocellular  layer  which  extends  inward 
from  the  surface  of  the  lobe  (Fig.  3B). 

Primary  afferent  fibers.  The  morphology  of  the  primary  afferent  fibers  is  re- 
vealed clearly  by  ganglionic  injections  of  peroxidase.  The  nerve  roots  enter  the 
spinal  cord  from  its  lateral  aspect;  in  the  case  of  lobe  4,  the  root  has  a  slight  rostral 
inclination  as  it  penetrates  the  cord.  A  few  root  fibers  terminate  in  the  dorsal  horn 
beneath  the  accessory  lobes.  The  vast  majority  of  the  primary  afferent  fibers  enter 
the  accessory  lobes  from  below  and  turn  radially  outward  to  terminate  throughout 
the  substance  of  the  lobe.  A  given  primary  afferent  fiber  may  branch  repeatedly 
in  its  course  outward  through  the  lobe.  Numerous  terminal  swellings  occur  through- 
out the  lobe,  however  a  heavier  band  of  terminal  arborization  appears  in  the  outer 
50  /^m  of  the  neuropil  of  the  lobe,  i.e.  immediately  subjacent  to  the  superficial 
parvocellular  layer  (Fig.  4A).  A  few  terminal  swellings  are  scattered  amongst  the 
somata  in  the  parvocellular  layer  (Fig.  4B),  but  the  bulk  of  the  terminals  and  en 
passant  swellings  occur  in  the  subjacent  neuropil.  This  pattern  of  termination  occurs 
in  all  the  accessory  lobes,  minor  as  well  as  major. 

Somatotopic  organization.  The  central  area  of  termination  of  each  fin  or  fin 
ray  nerve  was  determined  by  relying  on  transganglionic  transport  of  HRP.  This 
relatively  fine,  light  labeling  was  revealed  best  by  the  TMB  technique  and  darkfield 
microscopy  (Fig.  5 A)  although  the  reaction  product  was  clearly  visible  following 
reaction  with  the  modified  HY  substrate. 


SEA  ROBIN  SPINAL  CORD 


159 


FIGURE  5.  Horizontal  section  through  the  rostral  spinal  cord  of  a  sea  robin,  anterior  upward.  (A) 
Darkfield  photomicrograph  from  a  case  in  which  the  dorsal  fin  ray  nerve  had  been  injected  on  the  left 
side  and  the  ventral  fin  ray  nerve  had  been  injected  on  the  right  side.  Transganglionic  transport  of  the 
peroxidase  tracer  shows  in  the  photograph  as  a  bright  area  (lobe  3  on  the  left  and  lobe  1  on  the  right). 
In  these  cases,  no  label  appears  in  lobe  2  or  the  minor  accessory  lobe,  above  lobe  3.  (B)  Semischematic 
drawing  of  Fig.  5A  showing  the  somatotopic  representation  of  the  various  nerves  in  the  rostral  spinal 
cord.  1-4,  accessory  lobes;  ND,  dorsal  fin  ray  nerve;  NM,  middle  fin  ray  nerve;  NPFi,  inferior  pectoral 
fin  nerve;  NPFs,  superior  pectoral  fin  nerve;  NV,  ventral  fin  ray  nerve.  Same  scale  as  Fig.  5A. 

Application  of  HRP  to  the  nerve  innervating  the  ventral  fin  ray  resulted  in 
labeling  of  terminals  in  lobe  1,  the  middle  fin  ray  in  lobe  2,  and  the  dorsal  fin  ray 
in  lobe  3  (Fig.  5).  There  was  virtually  no  overlap  between  nerves  in  their  projection 
onto  the  accessory  lobes.  The  pectoral  fin  nerves  terminate  in  the  minor  accessory 
lobe  (lobe  4).  The  terminals  from  the  inferior  nerve  occupy  the  caudolateral  one- 
quarter  of  the  lobe  with  the  superior  nerve  terminals  filling  the  remaining  three- 
quarters  of  the  structure  (Fig.  5B).  In  summary,  the  central  representation  of  the 
fin  rays  and  pectoral  fin  is  somatotopically  organized.  The  ventral  fin  ray,  farthest 
from  the  fin,  is  represented  most  posteriorly  and  the  fin  itself  most  anteriorly. 

No  primary  afferent  fibers  ascend  in  the  cord  to  reach  the  level  of  the  caudal 
medulla.  Therefore  there  does  not  appear  to  be  a  system  in  this  species  homologous 
to  the  dorsal  columns  found  in  amniote  vertebrates. 


DISCUSSION 

Sea  robins  use  their  free  fin  rays  to  explore  their  surroundings.  That  the  fin 
rays  are  chemoreceptive  has  been  demonstrated  both  by  behavioral  and  electro- 
physiological  means  (Scarrer  et  al.,  1947;  Bardach  and  Case,  1965).  Since  the  fin 
rays  lack  taste  buds  and  are  innervated  only  by  spinal  nerves,  this  chemosensitivity 
has  been  attributed  to  the  common  chemical  sense  (Parker,  1922).  Therefore  at 


160  THOMAS  E.  FINGER 

least  some,  if  not  most,  of  the  fibers  in  the  fin  ray  nerves  mediate  the  common 
chemical  sensibility.  Compared  to  other  spinal  nerves,  the  pectoral  fin  and  fin  ray 
nerves  are  unique  in  terminating  in  the  accessory  lobes.  Accordingly,  the  accessory 
lobes  probably  are  involved  in  processing  input  from  the  common  chemical  sense. 

The  pectoral  fin  and  fin  rays  are  represented  in  a  somatotopic  order  in  the  spinal 
cord.  Somatotopy  in  a  chemosensory  system  is  not  unique  to  this  modality;  a  gus- 
tatory somatotopy  has  been  described  for  catfish  at  the  level  of  both  the  primary 
(Finger,  1976)  and  secondary  (Finger,  1978)  sensory  nuclei. 

One  surprising  result  in  this  study  is  the  order  of  the  somatotopic  map  in  the 
spinal  cord.  The  ventral  fin  ray,  which  is  also  the  most  anterior  part  of  the  fin,  is 
represented  in  the  most  posterior  accessory  lobe.  The  pectoral  fin,  which  lies  pos- 
terior (and  dorsal)  to  the  fin  rays  is  represented  farther  anteriorly  in  the  cord.  If 
the  pattern  of  innervation  reflects  the  dermatome  of  origin  in  the  embryo,  then  this 
implies  that  the  anteroventral  part  of  the  fin,  including  the  fin  rays,  arises  posterior 
to  the  rest  of  the  fin.  If  so,  this  further  implies  that  the  fin  has  rotated  during 
embryogenesis  such  that  the  posterior  edge  of  the  fin  in  the  embryo  moves  ventrally 
and  rostrally  during  development  so  as  to  lie  anterior  and  ventral  to  the  rest  of  the 
fin  in  the  adult. 

The  description  of  the  accessory  lobes  given  in  this  study  is  not  identical  to  the 
descriptions  offered  by  Morrill  (1895),  Ussow  (cited  in  Morrill,  1895),  or  Herrick 
(1907).  These  authors  describe  six  accessory  lobes;  the  present  report  describes 
four.  This  difference  is  attributable  to  the  more  detailed  study  of  intrinsic  mor- 
phology and  connections  given  in  the  present  report.  Four  morphologically  similar 
accessory  lobes  are  described  herein  and  they  correspond  to  the  accessory  lobes  2- 
6  of  Herrick  (1907)  which  are  identical  to  the  five  caudal  lobes  (unnamed)  illus- 
trated by  Morrill  (1895).  Both  of  these  authors  divide  accessory  lobe  2  of  this  study 
into  two  parts  on  the  basis  of  the  sulcus  in  which  the  lobar  blood  vessel  lies  (see 
above).  However,  upon  careful  examination  (see  Fig.  2),  all  the  major  accessory 
lobes  are  marked  by  a  similar  sulcus.  Since  the  portions  of  each  lobe  rostral  and 
caudal  to  this  sulcus  are  indistinguishable  both  in  terms  of  morphology  and  con- 
nections, there  is  no  apparent  reason  to  separate  these  two  halves  of  the  same 
structure.  In  addition  to  the  doubling  of  this  central  major  accessory  lobe  (lobe  2), 
both  Herrick  and  Morrill  describe  another  accessory  lobe  lying  rostral  to  the  minor 
accessory  lobe  (lobe  4  of  this  study).  This  more  rostral  structure  receives  input 
predominantly  from  descending  primary  afferent  fibers  of  the  trigeminal  nerve  (T. 
Finger,  unpub.  obs. ).  As  such,  this  structure  is  similar  to  the  medial  funicular 
nucleus  described  by  Herrick  (1906)  and  Finger  (1976).  Furthermore  the  mor- 
phology of  this  medial  funicular  nucleus  in  sea  robins  (Herrick's  accessory  lobe  1) 
is  quite  different  from  that  of  the  accessory  lobes  proper.  The  medial  funicular 
nucleus  lacks  the  external  cell  layer  which  is  characteristic  of  the  accessory  lobes. 
Accordingly,  the  present  study  does  not  include  the  medial  funicular  nucleus  among 
the  spinal  accessory  lobes. 

The  minor  accessory  lobe  (lobe  4)  receives  input  from  the  nerves  innervating 
the  pectoral  fin  which  itself  is  supported  by  numerous  fin  rays.  One  possible  ex- 
planation for  the  lobules  in  the  minor  accessory  lobe  is  that  each  fin  ray  of  the 
pectoral  fin  is  represented  in  a  single  lobule.  This  conjecture  needs  to  be  tested  by 
either  finer  anatomical  or  electrophysiological  experiments.  Furthermore,  since  the 
morphology  of  the  minor  accessory  lobe  is  similar  to  the  major  lobes,  the  pectoral 
fin  itself  may  be  capable  of  chemoreception  albeit  with  less  sensitivity  or  discrim- 
inability  than  the  free  fin  rays.  Whitear  (1971)  reports  that  isolated  chemosensory 


SEA  ROBIN  SPINAL  CORD  161 

cells  are  scattered  throughout  the  epidermis  of  many,  if  not  all,  teleosts.  Accord- 
ingly, the  fin  ray  chemosense  may  represent  a  specialization  of  a  spinal  chemosense 
present  in  many  vertebrates  (Parker,  1922). 

LITERATURE  CITED 

BARDACH,  J.  E.,  ANoJ.  CASE.  1965.  Sensory  capabilities  of  the  modified  fins  of  squirrel  hake  (Urophycus 

chuss)  and  searobins  (Prionotus  carolinus  and  P.  evolans).  Copeia  1965:  194-206. 
BELL,  C.  C,  T.  E.  FINGER,  AND  C.  RUSSELL.  1981.  Central  connections  of  the  posterior  lateral  line 

lobe  in  mormyrid  fish.  Exp.  Brain  Res.  42:  9-22. 
FINGER,  T.  E.  1 976.  Gustatory  pathways  in  the  bullhead  catfish.  I.  Connections  of  the  anterior  ganglion. 

J.  Comp.  Neurol.  165:  513-526. 
FINGER,  T.  E.  1978.  Gustatory  pathways  in  the  bullhead  catfish.  II.  Facial  lobe  connections.  J.  Comp. 

Neurol.  180:  691-706. 
HERRICK,  C.  J.  1906.  On  the  centers  for  taste  and  touch  in  the  medulla  oblongata  of  fishes.  /.  Comp. 

Neurol.  16:  403-440. 
HERRICK,  C.  J.  1907.  The  tactile  centers  in  the  spinal  cord  and  brain  of  the  sea  robin  Prionotus  carolinus 

L.  J.  Comp.  Neurol.  17:  307-327. 

MESULAM,  M.  M.  1978.  Tetramethyl  benzidine  for  horseradish  peroxidase  neurohistochemistry.  A  non- 
carcinogenic  blue  reaction-product  with  superior  sensitivity  for  visualizing  neural  afferents  and 

efferents.  J.  Histochem.  Cytochem.  26:  106-117. 

PARKER,  G.  H.  1922.  Smell,  Taste  And  Allied  Senses  In  The  Vertebrates.  Lippincott,  Philadelphia. 
SCHARRER,  E.,  S.  W.  SMITH,  ANDS.  L.  PALAY.  1947.  Chemical  sense  and  taste  in  the  fishes  Prionotus 

and  Trichogaster.  J.  Comp.  Neurol.  86:  183-1981. 
WHITEAR,  M.  1971.  Cell  specialization  and  sensory  function  in  fish  epidermis.  J.  Zool.  Land.  163:  237- 

264. 


Reference:  Biol.  Bull.  163:  162-171.  (August,  1982) 


MORPHOLOGICAL  AND  BEHAVIORAL  IDENTIFICATION  OF  THE 

SENSORY  STRUCTURES  MEDIATING  PHEROMONE  RECEPTION 

IN  THE  BLUE  CRAB,  CALLINECTES  SAPIDUS 

RICHARD  A.  GLEESON 

Monell  Chemical  Senses  Center,  3500  Market  St..  Philadelphia,  PA  19104,  and  *  Whitney  Marine 
Laboratory,  University  of  Florida,  Route  I,  Box  121,  St.  Augustine,  FL  32084 

ABSTRACT 

Scanning  electron  microscopy  was  used  to  survey  the  aesthetasc  tuft  on  the 
outer  flagellum  of  the  antennule  (1st  antenna)  in  order  to  identify  sensilla  poten- 
tially involved  in  pheromone  detection  by  the  male  blue  crab.  These  studies  showed 
that  the  tuft  of  each  antennule  is  divided  into  a  mesial  and  lateral  half  by  a  region 
of  cuticle  from  which  no  sensilla  arise.  Two  setal  types  were  revealed:  the  aesthetascs 
and  previously  undescribed  sensilla  which  originate  exclusively  on  the  mesial  side 
of  the  tuft  and  project  to  the  lateral  half  between  the  rows  of  aesthetascs.  Exper- 
iments were  performed  in  which  the  mesial  half,  lateral  half,  or  entire  aesthetasc 
tuft  was  bilaterally  ablated  from  the  antennules  of  test  males.  As  revealed  by 
behavioral  tests,  pheromone  responses  in  "mesial  half1  and  "lateral  half  ablation 
groups  were  reduced  22%  and  21%,  respectively,  relative  to  control  (P  >  0. 10); 
whereas  a  highly  significant  (P  <  0.005)  response  decrement  (80%  relative  to  con- 
trol) occurred  in  the  "entire  tuft"  ablated  group.  The  data  suggest  that  pheromone 
reception  in  the  male  blue  crab  is  effected  via  the  aesthetascs.  The  relationship  of 
these  findings  to  those  for  other  decapod  crustaceans  is  discussed. 

INTRODUCTION 

Previous  work  demonstrated  the  presence  of  a  pheromone  in  the  urine  of  pu- 
bertal  females  of  C.  sapidus  which  triggers  courtship  behavior  in  males  (Gleeson, 
1980).  It  was  further  shown  that  detection  of  this  pheromone  occurs  via  chemo- 
receptors  located  on  the  outer  flagellum  of  the  antennules  (first  antennae)  as  in- 
dicated by  the  lack  of  courtship  responses  for  males  in  which  the  outer  flagella  were 
ablated. 

In  the  present  study  the  outer  flagellum  was  examined  using  scanning  electron 
microscopy  (SEM)  to  identify  structures  potentially  involved  in  pheromone  recep- 
tion. This  effort  focused  on  the  aesthetasc  tuft  region  since  these  sensilla  are  pre- 
sumed to  be  chemosensory  ("olfactory  receptors")  in  decapods  (Ache,  in  press). 
Based  on  the  morphological  information,  various  lesions  were  made  in  the  tuft  and 
any  decrement  in  pheromone  response  was  assessed  behaviorally. 

MATERIALS  AND  METHODS 
Morphology 

Antennules  were  treated  in  Karnovsky's  fixative  for  two  to  two  and  a  half  hours, 
rinsed  in  0. 1  M  sodium  cacodylate  buffer,  and  dehydrated  through  a  graded  ethanol 

Received  26  January  1982;  accepted  21  May  1982. 
*  Present  address. 


162 


PHEROMONE  RECEPTION  IN  CALLINECTES  163 

series.  The  samples  were  then  transferred  to  acetone,  subjected  to  critical  point 
drying,  and,  after  gold  coating,  examined  with  a  scanning  electron  microscope. 

To  evaluate  the  permeability  of  structures  on  the  outer  flagellum,  the  crystal 
violet  technique  of  Slifer  (1960)  was  utilized.  Antennules  were  fixed  in  a  10% 
solution  of  formalin  for  24  hours,  then  rinsed  in  water  and  exposed  to  a  0.5% 
solution  of  crystal  violet  for  periods  varying  from  five  seconds  to  10  minutes.  After 
two  rinses  in  distilled  water  the  specimens  were  dried,  cleared  in  xylene,  and 
mounted  for  inspection  under  the  light  microscope. 

Ablation  experiments 

All  studies  were  performed  during  the  summer  months  using  the  facilities  at 
the  University  of  Maryland's  Marine  Products  Laboratory  in  Crisfield,  Maryland. 
Animals  were  obtained  locally  from  commercial  sources,  held  in  tanks  (1.2  X  2.4 
X  0.3  m)  with  a  flow-through  water  system,  and  sustained  on  a  diet  of  fish. 

A  test-tank  (1.0  X  1.0  X  0.2  m)  in  which  the  water  depth  was  maintained  at 
10  cm  via  a  standpipe  drain  was  used  for  all  behavioral  testing.  Water  filtered  to 
10  p,m  was  introduced  to  one  corner  of  the  tank  at  a  rate  of  approximately  five 
liters  per  minute.  As  a  source  of  pheromone,  three  to  six  pubertal  females  (those 
within  six  days  of  undergoing  their  maturity  molt)  were  retained  in  an  acrylic 
cylinder  (15  cm  height  by  30  cm  diameter)  which  was  positioned  close  to  the  inflow 
corner  of  the  test-tank. 

In  each  trial  six  male  crabs  were  introduced  to  the  test-tank  immediately  fol- 
lowing placement  of  the  females  within  the  cylinder.  The  activity  of  the  males  was 
observed  over  a  five  minute  period  after  which  the  position  of  the  inflow  delivery 
tube  was  switched  to  overflow  the  water  in  the  cylinder.  The  actions  of  the  males 
were  then  noted  over  a  second  five  minute  observation  period  and  courtship  re- 
sponses recorded  as  defined  previously  (Gleeson,  1980).  The  criteria  used  to  identify 
courtship  behavior  were: 

1 )  A  courtship  display — chelae  extended  in  the  lateral  position,  swimming  ap- 
pendages (fifth  pereopods)  rotated  anterodorsally  and  waved  from  side  to  side  above 
the  carapace,  and  walking  legs  (second  to  fourth  pereopods)  extended  such  that 
the  body  is  elevated  to  a  near  maximum  height  above  the  substrate;  or 

2)  An  approach  towards  another  test-male  with  chelae  extended  in  the  lateral 
position,  followed  by  an  attempt  to  cradle-carry  the  approached  individual. 

All  males  were  pre-tested  in  the  apparatus,  and  only  those  exhibiting  courtship 
responses  were  used  for  ablation  treatments.  These  treatments  involved  bilateral 
antennule  operations  performed  under  a  dissecting  microscope.  The  crabs  were 
restrained  and  each  antennule  held  in  position  by  a  clamp  mounted  on  a  micro- 
manipulator  which  allowed  positioning  the  antennule  such  that  the  aesthetasc  tuft 
was  accessible  for  ablation.  Four  treatment  categories  were  examined  using  ran- 
domly selected  males: 

1 )  Ablation  of  the  entire  aesthetasc  tuft.  Water  was  blotted  from  the  tuft  and 
the  hairs  manipulated  from  their  normally  recumbent  position  to  allow  cutting 
the  entire  tuft  with  micro-dissecting  scissors. 

2)  Ablation  of  the  mesial  portion  of  the  tuft.  Again,  this  involved  blotting  the 
tuft  and  manipulating  the  hairs  from  their  recumbent  position.  Fine-tipped 
forceps  were  used  to  pluck  all  of  the  hairs  from  the  mesial  half  of  the  tuft, 
leaving  the  aesthetascs  of  the  lateral  half  intact. 


164 


RICHARD  A.  GLEESON 


FIGURE  1.  Lateral  view  of  antennule  tip  showing  aesthetasc  tuft  (arrowhead)  on  outer  flagellum. 
Inner  flagellum  was  removed.  Scale  bar  =  700  ^m. 

3)  Ablation  of  the  lateral  portion  of  the  tuft.  The  procedure  was  as  in  (2)  except 
that  the  lateral  half  of  the  tuft  was  removed  and  the  mesial  portion  left  intact. 

4)  Sham  control.  This  process  was  as  for  all  of  the  above  treatments,  but  with 
no  cutting  or  plucking  of  hairs  within  the  tuft. 

Between  24  to  48  hours  after  ablation  treatments  the  males'  pheromone  re- 
sponses were  tested.  In  order  to  reduce  the  incidence  of  false  negatives  (e.g.,  lack 
of  response  due  to  the  stimulus  failing  to  reach  receptor  sites),  each  male  was 
examined  in  up  to  three  trials.  Two  untreated  control  males  were  simultaneously 
tested  with  treated  animals  in  every  trial,  and  any  trial  in  which  none  of  the  six 
males  responded  was  voided. 

At  the  conclusion  of  the  behavioral  tests,  the  antennules  of  the  treated  males 
were  removed  and  prepared  for  SEM  inspection. 

RESULTS 
Morphology 

The  outer  flagellum  of  the  antennule  (Fig.  1 )  is  approximately  2  mm  in  length 
and  characterized  by  a  series  of  over  30  segments  which  give  it  flexibility.  A  prom- 
inent feature  of  this  flagellum  is  the  tuft  of  approximately  650-700  aesthetasc  hairs 
which  arise  from  grooves  distally  situated  on  the  ventral  surface  of  most  flagellar 
segments  (Fig.  1  and  2A).  This  tuft  is  divided  into  mesial  and  lateral  halves  by  a 
central  region  of  cuticle  from  which  no  aesthetascs  arise  (Fig.  3A).  The  aesthetasc 
setae  are  from  700-1000  ^m  in  length  and  approximately  10-12  ^m  in  diameter. 
Three  to  five  distinct  bulges  (Fig.  3B)  are  characteristic  of  the  proximal  region  of 


PHEROMONE  RECEPTION  IN  CALLINECTES 


165 


FIGURE  2.  (A)  Ventro-lateral  view  of  tuft  region.  Lateral  half  of  tuft  was  removed  allowing 
visualization  of  the  asymmetric  sensilla  (arrowhead).  Aesthetascs  (broken)  in  groove  on  the  distal  border 
of  a  flagellar  segment  are  indicated  by  the  arrow.  Scale  bar  =  135  pm.  (B)  5X  magnification  of  the 
enclosed  region  in  (A).  Arrowhead  indicates  socket  location  of  an  asymmetric  sensillum  on  mesial  side 
of  tuft.  Scale  bar  =  27  p.m. 


166 


RICHARD  A.  GLEESON 


FIGURE  3.  (A)  Ventral  view  of  tuft  in  which  a  portion  of  the  mesial  half  was  removed.  Note  central 
region  of  cuticle  lacking  sensilla  (arrows).  Asymmetric  sensilla  indicated  by  arrowheads.  Scale  bar 
=  100  urn.  (B)  5X  magnification  of  the  enclosed  region  in  (A).  Arrow  indicates  socket  of  asymmetric 
sensillum.  Black  arrowheads  show  pore  structures  in  cuticle.  Annular  bulge  in  basal  region  of  aesthetasc 
indicated  by  black  on  white  arrowhead.  Scale  bar  =  20  nm. 


PHEROMONE  RECEPTION  IN  CALLINECTES  167 

each  aesthetasc,  and  these  give  way  to  periodic  annulations  (about  30  /mi  apart) 
for  the  remainder  of  the  hair  shaft  until  the  seta  tapers  to  a  tip,  approximately  2 
/mi  in  diameter,  which  lacks  a  terminal  pore. 

Confined  to  the  mesial  half  of  the  tuft,  and  proximal  to  the  aesthetasc  row  of 
each  segment,  are  groups  of  sensory  hairs  (0-4  per  flagellar  segment)  with  an 
external  morphology  unlike  that  of  the  aesthetascs.  These  setae,  herein  referred 
to  as  asymmetric  hairs  because  of  their  exclusively  mesial  origin,  arise  from  sockets 
which  project  from  the  cuticle  at  an  angle  such  that  the  hairs  extend  across  the 
tuft  from  the  mesial  to  the  lateral  side  (Fig.  2A,  B  and  3A,  B).  The  asymmetric 
setae  range  in  length  from  170  to  220  /mi,  with  diameters  between  6  and  8  /urn  at 
the  base,  tapering  gradually  to  a  1  /mi  tip  with  no  terminal  pore.  Numbers  of  these 
hairs  range  from  46  to  70  per  flagellum. 

The  only  other  surface  features  in  the  tuft  region  are  small  pores  (0.3-0.6  /mi 
in  diameter)  which  are  distributed  along  the  distal  portion  of  each  flagellar  segment 
just  proximal  to  the  groove  from  which  the  aesthetascs  arise  (Fig.  3B).  Accurate 
counts  of  these  structures  are  lacking,  but  the  numbers  range  on  the  order  of  20 
to  60  per  segment.  Observations  using  light  microscopy  revealed  that  the  pores  are 
openings  of  canals  extending  3-4  /mi  through  the  cuticle  from  spherical  chambers 
(approximately  3  /mi  in  diameter)  which  are  situated  on  the  inner  surface  of  the 
cuticle.  The  aesthetasc  tuft  is  the  only  region  of  the  antennule  in  which  these  pore 
structures  are  found. 

Permeability  studies  using  crystal  violet  showed  that  both  the  aesthetascs  and 
asymmetric  setae  were  penetrated  within  five  seconds.  The  asymmetric  hairs  were 
stained  along  their  entire  length,  whereas  a  differential  penetration  occurred  in  the 
aesthetascs.  The  basal  region  of  each  aesthetasc  (that  section  in  which  the  annular 
bulges  are  located)  was  less  darkly  stained  than  the  remaining  portion  of  the  hair, 
even  after  10  minutes  of  exposure. 

Concurrent  SEM  studies  using  antennules  from  females  revealed  no  obvious 
sexual  dimorphism  in  numbers  or  morphological  types  of  sensory  structures  on  the 
outer  flagellum. 

Ablation  experiments 

As  revealed  by  SEM,  more  than  90%  of  the  setae  in  the  targeted  regions  of  the 
aesthetasc  tuft  were  removed  or  otherwise  lesioned  in  nearly  all  experimental  an- 
imals (Fig.  4  and  5).  The  results  of  these  studies  are  graphically  depicted  in  Figure 
6.  Removal  of  the  mesial  and  lateral  portions  of  the  aesthetasc  tuft  produced  nearly 
equal  reductions  in  the  response  levels  of  males.  These  reductions  are  not  statis- 
tically significant,  however,  when  compared  to  the  control  group  using  a  Chi-square 
evaluation  (P  >  0.10).  In  contrast,  for  males  in  which  the  entire  tuft  was  ablated, 
the  response  decrement  is  highly  significant  (P  <  0.005)  when  compared  to  any 
of  the  other  treatment  categories.  These  latter  data  are  further  supported  by  an 
additional  group  of  seven  males  in  which  the  aesthetasc  tufts  were  similarly  ablated; 
all  were  unresponsive  to  pheromone  stimulation  when  examined  in  two  trials  each. 

DISCUSSION 

This  study  provides  evidence  which,  in  conjunction  with  previous  morphological, 
behavioral,  and  physiological  work,  corroborates  the  postulated  chemosensory  func- 
tion of  the  aesthetasc  setae  (see  for  example  Laverack,  1964;  Ghiradella  et  al., 
1968;  Hazlett,  1971 ).  Specifically,  the  data  establish  that  these  setae  are  of  critical 
importance  to  the  male  C.  sapidus  in  detecting  the  pheromone  of  the  pubertal 


168 


RICHARD  A.  GLEESON 


FIGURE  4.    Ventro-mesial  view  of  tuft  region  in  which  setae  were  cut  with  micro-dissecting  scissors. 
Scale  bar  =  200  ^m. 


FIGURE.  5.     Ventro-mesial  view  of  tuft  region  in  which  mesial  half  was  removed  using  forceps. 
Scale  bar  =  200  ^m. 


PHEROMONE  RECEPTION  IN  CALLINECTES 
lOOi 


169 


80 


60 


RESPONDING 


40 


20 


N=28 


=  33 


MESIAL     LATERAL     TOTAL     CONTROL 

FIGURE.  6.  Courtship  responses  of  males  in  which  the  mesial  half  (mesial),  lateral  half  (lateral), 
or  entire  (total)  aesthetasc  tuft  was  ablated  from  both  antennules.  Sham  control  (control)  procedure 
was  as  for  other  treatment  groups,  but  with  no  removal  of  setae.  N  =  number  of  animals  tested. 


female,  thus  extending  the  findings  of  earlier  experiments  which  localized  this 
detection  to  the  outer  flagellum  of  the  antennules  (Gleeson,  1980). 

Cutting  the  tuft  (entire  tuft  ablation  treatment)  reduced  the  length  of  the 
aesthetascs  such  that  less  than  one  third  remained  intact,  and  this  procedure  de- 
cidedly blocked  pheromone  detection  in  males  so  treated.  On  the  other  hand,  despite 
lesions  to  half  the  tuft,  the  animals  in  the  mesial  and  lateral  ablation  groups  retained 
their  ability  to  detect  the  pheromone  and  also  appeared  equally  competent  in  this 
detection  capability,  albeit  at  a  reduced  level  relative  to  control.  This  implies  that 
the  receptors  required  for  pheromone  recognition  are  common  to  both  halves  of 
the  tuft.  If  convergence  of  primary  sensory  neurons  onto  second  order  olfactory 
cells  is  involved  in  amplifying  a  pheromone  signal  (van  Drongelen  et  al.,  1978), 
the  reduced  response  level  in  these  treatment  groups  might  therefore  reflect  an 
increase  in  detection  threshold  resulting  from  loss  of  half  the  peripheral  input  to 
second  order  cells. 

SEM  inspection  of  the  aesthetasc  tuft  region  revealed  relatively  few  sensory 
structures  as  compared,  for  example,  to  Pagurus  (Snow,  1974)  and  Panulirus 
(Laverack,  1964).  Other  than  the  aesthetascs  the  only  setae  in  this  zone  are  the 
asymmetric  hairs  which  are  confined  to  the  mesial  half  of  the  tuft.  Their  removal 
clearly  did  not  alter  pheromone  detection  in  test  males  as  indicated  by  the  equally 
responsive  mesial  and  lateral  ablation  groups.  They  are  quite  permeable  to  crystal 
violet,  however,  suggesting  a  possible  chemoreceptive  role,  but  this  remains  to  be 
determined  physiologically.  Extracellular  recordings  from  axons  of  cells  innervating 
the  asymmetric  hairs  revealed  that  these  structures  are  at  least  mechanosensory. 
Deflection  of  the  hair  using  a  fine  glass  probe  elicits  phasic  bursts  of  action  potentials 
(Gleeson,  unpublished  data).  The  orientation  of  the  asymmetric  setae  (i.e.,  pro- 
jection across  the  tuft  between  the  rows  of  aesthetascs)  in  conjunction  with  this 
preliminary  physiological  data  suggests  that  these  structures  may  serve  to  monitor 
water  flow  through  the  tuft,  such  as  would  occur  during  flicking  of  the  antennule 
(Schmitt  and  Ache,  1979). 

The  significance  of  the  pores  located  exclusively  in  the  tuft  region  is  an  intriguing 
unknown.  Similar  structures  have  been  found  associated  with  the  aesthetascs  of 


170  RICHARD  A.  GLEESON 

Homarus  americanus  (Atema,  1977;  Derby,  1982)  and  with  certain  setal  types  on 
the  antennae  of  the  sergestid  shrimp,  Acetes  sibogae  australis  (Ball  and  Cowan, 
1977).  However,  since  data  on  the  underlying  structure  of  these  pores  is  incomplete, 
speculation  as  to  their  function  must  await  further  study. 

The  question  of  pheromone  receptor  location  has  been  addressed  at  various 
levels  in  other  crustaceans.  For  Palaemon  paucidens,  Kamiguchi  (1972)  reported 
that  the  inner  branch  of  the  bifurcated  outer  flagellum  of  the  antennule  is  relatively 
longer  in  males  than  in  females  and  has  a  greater  number  of  sensory  (presumably 
aesthetasc)  hairs.  Although  no  experiments  were  conducted  to  test  the  hypothesis, 
it  is  postulated  that  this  dimorphism  is  related  to  sex  pheromone  detection  on  the 
part  of  the  male  as  has  been  found  to  be  the  case  in  many  insect  species  (Schneider, 
1964).  A  similar  sexual  dimorphism  in  the  quantitative  distribution  of  aesthetascs 
has  been  reported  for  several  other  crustacean  groups  as  well  (Barber,  1961). 

Christofferson  (1970)  noted  that  removal  of  the  aesthetasc-bearing  outer  fla- 
gellum [erroneously  labeled  the  inner  flagellum  in  that  study  and  uncorrected  in 
Dunham's  (1978)  review]  from  the  antennulesof  the  male  Portunus  sanguinolentus 
blocked  the  behavioral  response  to  the  female's  sex  pheromone.  This  response  was 
not  affected  in  control  animals  in  which  the  inner  flagellum  (mislabeled  the  outer 
flagellum)  was  removed. 

Based  on  ablation  and  electroantennulogram  studies,  Ameyaw-Akumfi  and 
Hazlett  (1975)  and  Ameyaw-Akumfi  (1976)  concluded  that  the  inner  (non-aes- 
thetasc  bearing)  flagellum  of  the  antennule  in  the  male  crayfish,  Procambarus 
clarkii,  contains  chemoreceptors  mediating  sex  recognition.  The  evidence  on  which 
this  conclusion  is  based,  however,  is  not  entirely  convincing.  Although  it  is  stated 
that  test  animals  were  unresponsive  following  removal  of  the  inner  flagellum,  no 
data  are  presented  for  evaluation.  Furthermore  an  important  control  condition  is 
lacking:  namely,  an  examination  of  test  animals  following  ablation  of  the  outer 
flagellum.  Since  the  physiological  data  do  not  contribute  to  a  resolution  of  this 
issue,  the  potential  role  of  the  aesthetascs  in  sex  recognition  by  P.  clarkii  remains 
uncertain.  Indeed,  the  situation  is  further  confounded  by  the  experiments  of  Itagaki 
and  Thorp  (1981)  who  used  a  flow-through  design  to  examine  chemical  commu- 
nication in  P.  clarkii  and  found  no  evidence  for  chemically  mediated  sex  recognition 
in  this  species. 

Dahl  el  al.,  (1970a,  b)  present  evidence  suggesting  pheromone  reception  in 
Gammarus  duebeni  occurs  via  calceoli  which  are  male-specific  sensory  structures 
located  on  the  second  antennae.  Their  hypothesis  is  based  on  the  apparent  binding 
of  a  female-specific  natural  product  to  the  calceoli,  as  demonstrated  in  males  ex- 
posed to  water  in  which  radiolabeled  females  were  retained.  Recent  work  by  Lyes 
(1979)  has  supported  this  hypothesis:  "masking"  or  ablating  the  second  antennae 
of  the  male  G.  duebeni  forestalls  pairing  with  females.  However,  Hartnoll  and 
Smith  (1980)  found  it  necessary  to  remove  both  the  first  and  second  antennae  to 
significantly  block  pairing;  indicating  that  recognition  of  premolt  females  can  be 
mediated  via  sensory  structures  other  than  the  calceoli. 

In  summary,  the  information  to  date  identifying  structures  mediating  phero- 
mone reception  in  decapod  crustaceans  is  fairly  limited  and  in  some  cases  requires 
further  experimentation.  The  present  study  implicates  the  aesthetascs  as  important 
receptors  for  sex  pheromone  detection  in  C.  sapidus,  but  whether  these  structures 
prove  to  generally  function  in  this  capacity  for  decapods  must  await  future  com- 
parative work  specifically  addressing  the  role  of  these  setae  in  sex  recognition. 


PHEROMONE  RECEPTION  IN  CALLINECTES  171 

ACKNOWLEDGMENTS 

I  wish  to  express  my  appreciation  to  Drs.  B.  Ache,  C.  Derby,  and  K.  Hamilton 
for  their  helpful  comments  during  the  preparation  of  the  manuscript.  I  am  also 
indebted  to  Dr.  R.  Lavker  for  his  assistance  in  the  SEM  phase  of  this  study,  and 
to  the  staff  at  the  University  of  Maryland's  Marine  Products  Laboratory,  partic- 
ularly Mr.  M.  Paparella,  for  their  support  during  the  behavioral  work.  A  special 
thanks  is  due  Mr.  T.  Long  for  his  assistance  in  supplying  experimental  animals. 
Research  support  was  provided  in  part  by  a  fellowship  grant  from  NIH 
#5F32NSO5942-02. 

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NEW  VICTORELLIDS  (BRYOZOA,  CTENOSTOMATA)  FROM 

NORTH  AMERICA:  THE  USE  OF  PARALLEL  CULTURES 

IN  BRYOZOAN  TAXONOMY 

DIETHARDT  JEBRAM1  AND  BETTY  EVERITT2 

1  Zoologisches  Institut  der  Technischen  Universitat  Braunschweig,  Pockelsstrasse  Wa,  D-3300 
Braunschweig,  Germany  (FRG);  and  2  Department  of  Zoology,  Louisiana  State  University, 

Baton  Rouge,  LA  70803 

ABSTRACT 

Three  species  of  the  Victorellidae  (Bryozoa,  Ctenostomata)  were  found  in  North 
America  and  were  cultivated  in  Braunschweig  ( W.  Germany).  Two  are  new  species, 
Victorella  pseudoarachnidia  sp.  nov.  and  Tanganella  appendiculata  sp.  nov.  Bul- 
bella  abscondita  Braem  is  reported  for  the  first  time  from  Massachusetts 
(U.  S.  A.).  Identification  of  victorellids  requires  the  examination  of  living  animals. 
The  species  considered  here  were  identified  by  observation  of  live  material  collected 
in  North  America  and  Germany  and  of  living  individuals  cultured  from  that  ma- 
terial. 

INTRODUCTION 

Kent  (1870)  described  a  species  of  the  bryozoa  (class  Gymnolaemata,  order 
Ctenostomata)  as  Victorella  pavida,  based  on  material  collected  in  brackish  waters 
in  England.  Kraepelin  (1887)  concluded  that  some  specimens  interpreted  primarily 
as  immature  V.  pavida  (from  the  Ryck  River,  Germany)  represented  a  new  species 
and  named  it  Paludicella  miilleri.  Braem's  (1911)  first  opinion  was  that  P.  miilleri 
was  only  a  developmental  stage  of  V.  pavida. 

After  many  years  of  observation  of  living  victorellids  from  northern  Germany, 
Braem  (1951)  split  the  literature  species  Victorella  pavida  into  three  species.  After 
a  comparison  with  preserved  V.  pavida  from  England,  Braem  designated  certain 
specimens  to  be  V.  pavida,  characterized  by  the  seasonal  production  of  an  inter- 
tentacular  tube,  the  absence  of  brooded  embryos,  and  the  location  of  the  cardiac 
sphincter  far  above  the  central  stomach.  Victorellids  which  produced  no  inter- 
tentacular  tube,  brooded  embryos  internally,  and  possessed  a  cardiac  sphincter  near 
the  central  stomach  were  placed  into  a  new  genus  as  Tanganella  miilleri.  Specimens 
characterized  by  shorter  peristomial  tubes,  a  small  intertentacular  tube,  external 
brooding  of  embryos,  and  the  cardiac  sphincter  moderately  above  the  central  stom- 
ach were  assigned  to  a  new  genus  and  new  species,  Bulbella  abscondita. 

Although  these  distinguishing  characteristics  were  adequately  described  by 
Braem  (1951),  Brattstrom  (1954)  expressed  some  doubt  about  Braem's  splitting 
of  V.  pavida.  Brattstrom  apparently  overlooked  the  important  fact  that,  unlike 
Braem,  most  workers  described  new  species  from  preserved  specimens.  Preserved 
ctenostomes,  however,  do  not  usually  exhibit  all  features  necessary  for  identifi- 
cation. The  characteristics  described  by  Braem  (1951)  have  been  confirmed  on 
living  specimens  from  other  localities  (Jebram,  1969,  1976).  These  studies  on  living 
victorellids,  then,  suggest  that  many  specimens  identified  from  preserved  material 
may  have  been  other  species. 

Received  4  November  1981;  accepted  24  May  1982. 

172 


VICTORELLIDAE  FROM  N.  AMERICA  173 

Another  facet  of  the  taxonomic  problem  in  the  Victorellidae  is  evident  in  several 
North  American  reports.  Osburn  (1944)  studied  Victorella  pavida  from  the  Ches- 
apeake Bay  (Atlantic  Coast,  U.  S.  A.)  and  reported  that  the  colonies  collected 
from  the  upper  (less  saline)  end  of  the  bay  tended  to  be  less  branched.  However, 
his  photograph  (1944,  PI.  V)  of  V.  pavida  shows  brooded  embryos  and  therefore 
suggests,  according  to  Braem's  (1951)  work,  that  at  least  a  part  of  Osburn's  material 
was  not  Victorella. 

Rogick  (1949)  described  Nolella  blakei  from  Woods  Hole,  Massachusetts 
(Atlantic  Coast,  U.  S.  A.)  and  compared  it  to  other  ctenostomes.  Soule  (1957) 
identified  specimens  from  the  Salton  Sea  (California,  U.  S.  A.)  as  TV.  blakei  and 
V.  pavida.  He  incorrectly  synonymized  Tanganella  miilleri  with  V.  pavida,  but  did 
not  discuss  the  major  distinguishing  characteristics  used  by  Braem  (1951).  Rogick 
(Soule,  1957)  confirmed  Soule's  identification  of  the  preserved  material.  Exami- 
nation of  Soule's  Salton  Sea  specimens  revealed  victorellids  but  no  Nolella  (Jebram, 
personal  observations,  1977). 

Hyman  ( 1959)  was  one  of  the  few  zoologists  who  acknowledged  Braem's  (1951) 
results,  but  she  misinterpreted  his  comments  on  a  crucial  point.  She  (1959,  p.  333) 
wrote  that  "brooding  arrangements  vary  greatly,  even  within  the  same  genus 
.  .  .",  and  she  misquoted  Braem  in  her  statement  that  in  V.  pavida  "each  egg  as 
it  emerges  from  the  supraneural  pore  is  caught  in  a  depression  of  the  adjacent 
dorsal  body  wall,  and  eventually  passes  into  the  coelom  of  the  dorsal  side  of  the 
vestibule  where  three  or  four  developing  embryos  may  be  found."  These  comments 
and  her  figures  125C  and  125D  apply  not  to  V.  pavida  but  to  T.  miilleri.  Victorella 
pavida  produces  an  intertentacular  tube  (not  a  simple  pore)  and  does  not  brood 
embryos  (Braem,  1951;  Jebram,  1969,  1976). 

Subsequent  investigators  essentially  ignored  Braem's  (1951)  findings  and  con- 
tinued to  use  predominantly  external  characteristics  for  the  identification  of  the 
victorellids  (Prenant  and  Bobin,  1956;  Sacchi  and  Carrada,  1962;  Carrada  and 
Sacchi,  1964;  Everitt,  1975;  Poirrier  and  Mulino,  1977).  Osburn  (1944)  and  Soule 
(1957)  synonymized  various  forms  with  V.  pavida.  Osburn  (1944)  and  Everitt 
(1975)  independently  suggested  that  branching,  the  formation  of  adventitious 
zooids  (another  external  characteristic),  correlates  positively  with  salinity.  Poirrier 
and  Mulino  (1977)  confirmed  this  correlation  in  49  of  52  samples  but  suggested 
that  "other  factors  .  .  .  may  also  influence  branching." 

The  confusion  evident  in  taxonomic  and  ecological  papers  on  Victorella  pavida, 
the  relatively  poor  condition  of  V.  pavida  specimens  in  the  British  Museum  (Jebram, 
Everitt,  personal  observations,  1980),  and  the  apparent  lack  of  any  specimens  of 
Nolella  blakei,  including  Rogick's  material  from  Woods  Hole  (Everitt),  led  to  the 
current  investigation.  During  the  fourth  conference  of  the  International  Bryozoo- 
logical  Association  in  Woods  Hole,  Massachusetts  (U.  S.  A.)  in  September  of  1977, 
we  discussed  some  problems  in  victorellid  taxonomy.  The  purposes  of  this  inves- 
tigation were  to  collect  N.  blakei  from  the  type  locality,  to  collect  victorellids  from 
the  Salton  Sea  (California,  U.  S.  A.),  to  culture  any  victorellids  collected,  and  to 
revise  the  taxonomy  of  these  North  American  victorellids  on  the  basis  of  obser- 
vations of  living  animals. 

MATERIALS  AND  METHODS 

In  September  of  1977  and  in  August  of  1978,  victorellids  were  collected  from 
the  bridge  pilings  at  the  outlet  of  Lagoon  Pond  on  Martha's  Vineyard  Island  (Mass., 
U.  S.  A.),  the  type  locality  for  Nolella  blakei.  Living  colonies  were  observed  and 


174  D.  JEBRAM  AND  B.  EVERITT 

were  either  preserved  in  10%  formalin  or  cultured  in  the  laboratory  (Braunschweig, 
W.  Germany).  In  October  of  1977,  victorellids  were  collected  in  the  Salton  Sea 
(California,  U.  S.  A.)  and  were  treated  likewise. 

The  methods  for  culturing  bryozoans  were  described  previously  (Jebram,  1977a, 
1977b,  1979).  The  methods  for  culturing  victorellids  were  developed  anew  according 
to  earlier  results  with  other  bryozoan  species  (Jebram,  1980b).  For  comparative 
morphology  the  victorellids  were  maintained  at  21-23°C  and  14-16%o  salinity  and 
were  fed  the  food  mixture  J5b,  the  composition  of  which  was  determined  experi- 
mentally. The  descriptions  of  the  species  provided  here  are  based  on  specimens 
cultivated  under  these  conditions  (Jebram,  1980b).  Details  on  morphology  and 
techniques  are  given  under  "Experimental  Biology". 

After  the  animals  attained  sexual  maturity,  they  were  compared  with  living 
victorellids  collected  in  Germany  and  with  earlier  descriptions  based  on  living 
specimens.  Cultures  of  Victorella  pavida,  Tanganella  mulleri,  and  Bulbella  abs- 
condita  have  been  maintained  in  the  laboratory  of  the  senior  author  (T.  mulleri 
since  1968).  Colonies  subcultured  from  the  holotype  specimens  of  the  American 
victorellids  discussed  here  are  being  maintained  for  further  study;  Louisiana 
(U.  S.  A.)  specimens  are  also  being  cultured  in  Braunschweig. 

Drawings  of  the  species  described  here  were  prepared  by  Jebram  from  pho- 
tographs of  living  animals.  Paratype  specimens  are  in  the  collections  of  the  authors. 

SYSTEMATIC  TREATMENT  AND  RESULTS 

Victorella  pseudoarachnidia  sp.  nov. 

Holotype  material:  Collection  of  D.  Jebram,  1978-10-10-1. 

Paratype  material:  U.  S.  National  Museum  of  Natural  History,  Smithsonian 
Institution  (Washington,  D.  C),  Cat.  No.  36,  USNM  No.  292472;  Bryozoan  Col- 
lections of  the  Allen  Hancock  Foundation,  Univ.  of  Southern  California  at  Los 
Angeles,  No.  185.1;  British  Museum  (Natural  History),  London;  personal  collec- 
tions of  authors. 

Name:  The  cystid  appendages  suggest  superficial  similarities  to  an  arachnidioid 
form. 

Synonyms:  "Victorella  pavida  Kent"  and  "Nolella  blakei  Rogick"  sensu  Soule, 
1957  (nee.  Victorella  pavida  Kent,  1870,  sensu  Braem,  1951;  nee  Nolella  blakei 
Rogick,  1949). 

Type  locality:  Salton  Sea,  California,  U.  S.  A. 

Description:  The  colonies  of  Victorella  pseudoarachnidia  are  composed  of  se- 
rially arranged  zooids  (Fig.  1).  Each  zooid  usually  produces  one  distal  and  two 
lateral  daughter  zooids  at  the  sides  of  the  basal  part  of  the  cystid.  The  branching 
pattern  of  these  daughter  zooids  is  regular  and  symmetrical.  Older  zooids  can 
produce  adventive  zooids  by  forming  "high  buds"  ("Hochknospen")  on  the  anal 
or  lateral  sides  of  the  peristomial  tube  (Fig.  2).  In  addition  to  the  encrusting  colony 
parts  ("forma  encrustans"  sensu  Braem,  1951)  are  often  free,  non-encrusting 
branches  ("forma  ascendens"  sensu  Braem,  1951)  of  zooid  series  formed  at  the 
borders  of  the  substrate  pieces  or  originating  from  the  high  buds.  The  colony  in 
this  species  thus  has  a  habit  somewhat  similar  to  that  of  most  other  victorellids. 

Each  zooid  is  composed  externally  of  two  main  parts,  a  basal  proximal  part 
(usually  encrusting  the  substrate)  and  an  upright  peristomial  tube.  The  basal  part 
is  broad  at  its  distal  end  and  narrow  proximally;  the  proximal  narrowing  usually 
is  relatively  abrupt. 

Encrusting  zooids  of  V.  pseudoarachnidia  have  cystid  appendages  which  usually 
originate  on  each  side  of  the  budding  sites  of  the  daughter  zooids.  The  basic  pattern 


VICTORELLIDAE  FROM  N.  AMERICA 


175 


Victorella   pseudoarachnidia 


FIGURES  1  -4:  Victorella  pseudoarachnidia  sp.  nov.  ( 1 )  part  of  the  holotype  colony  demonstrating 
the  budding  pattern  in  an  encrusting  colony;  (2)  "high  buds"  and  adventive  zooids  in  lateral  view;  (3) 
three  zooids  in  basal  view  demonstrating  the  budding  pattern  and  the  cystid  appendages;  (4)  sexually 
mature  zooid  in  lateral  view;  an,  anus;  cae,  caecum;  cap,  cystid  appendages;  car,  cardia;  car-sph,  cardiac 
sphincter;  col,  collar;  cst,  central  stomach;  it,  intertentacular  tube;  ov,  ovary;  pha,  pharynx;  pyl,  pylorus; 
rec,  rectum;  ten,  tentacles;  tst,  testis;  scale  bars  represent  1  mm. 

consists  of  9-10  sites  for  the  potential  formation  of  cystid  appendages  (Figs.  3,  11). 
All  cystid  appendages  may  branch  weakly  into  two  to  several  tips.  One  or  a  few 
of  the  distinct  appendages  may  be  vestigial  or  absent.  The  branching  of  the  ap- 


176  D.  JEBRAM  AND  B.  EVERITT 

pendages  may  occur  under  the  central  basal  part  of  the  cystid  so  that  only  the  tips 
project  visibly  beyond  the  lateral  margin  of  the  cystids.  The  non-encrusting  zooids 
form  no  (or  vestigial)  cystid  appendages.  The  formation  of  cystid  appendages  is 
generally  variable  and  somewhat  modifiable  but  never  occurs  on  the  narrow,  most 
proximal  part  of  the  basal  cystid.  The  origin  of  all  the  tips  of  the  cystid  appendages 
from  restricted  and  rather  distinct  sites  of  a  zooid  can  often  be  detected  only  if  the 
animals  are  growing  on  glass  slides  and  are  illuminated  sufficiently.  Otherwise  the 
shape  of  the  central  basal  cystid  parts  may  superficially  resemble  an  arachnidioid 
form.  In  the  victorellids,  the  appendages  of  different  zooids  may  touch  each  other 
but  never  really  fuse  histologically  as  they  can  in  typical  arachnidioid  forms. 

The  peristomial  tubes  grow  stepwise  upward  by  each  case  of  polypide  replace- 
ment and  may  thus  attain  a  considerable  length  (to  about  1  cm  with  polypide 
retracted).  The  diameter  of  the  peristomial  tube  is  about  220  j*m. 

The  autozooids  of  Victorella  pseudoarachnidia  always  have  8  tentacles.  Sex- 
ually mature  zooids  have  a  trumpet-shaped  intertentacular  tube  with  a  widely  flared 
opening,  through  which  the  ova  are  released  (Fig.  4).  This  species  does  not  brood 
embryos,  either  in  the  neck  region  or  in  the  tentacle  sheath.  Freshly  released  ova 
are  spindle-shaped  and  whitish  and  become  globular  after  approximately  fifteen 
minutes. 

The  gut  anatomy  of  this  species  shares  two  essential  attributes  with  that  of 
Victorella  pavida:  location  of  the  cardiac  sphincter  considerably  above  the  central 
stomach  (Fig.  4)  and  absence  of  a  gizzard. 

Victorella  pseudoarachnidia  produces  lasting  buds  which  may  have  irregularly 
shaped  marginal  appendages  and  which,  in  older  stages,  have  a  greyish  or  dark 
brown  to  almost  black  cuticle.  The  storage  products  in  the  ova  and  in  the  lasting 
buds  are  whitish. 

The  accompanying  fauna  in  the  type  locality  of  V.  pseudoarachnidia  included 
the  stolonate  ctenostome  Bowerbankia  cf.  gracilis  Leidy  and  the  kamptozoan  (en- 
toproct)  Barentsia  benedeni  (Foettinger)  (Jebram,  field  observations,  1977).  Soule 
(1957)  did  not  report  these  species  from  his  Salton  Sea  samples. 

Differentiating  characteristics  of  similar  forms:  ( 1 )  In  other  species  of  Victorella 
described  so  far,  there  has  been  no  mention  of  the  formation  of  such  types  of 
branched  cystid  appendages  at  restricted  sites  of  the  zooids.  (2)  Species  of  Tan- 
ganella  differ  in  the  location  of  the  cardiac  sphincter  near  the  central  stomach,  the 
formation  of  an  intertentacular  pore,  the  brooding  of  embryos,  and  the  form  and 
arrangement  of  the  cystid  appendages.  (3)  Almost  all  species  of  the  superfamily 
Arachnidioidea  have  the  potential  to  produce  cystid  appendages,  but  these  ap- 
pendages are  usually  narrow  and  rarely  branched  and  may  originate  also  from  the 
narrow  proximal  part  of  the  cystid.  In  the  Arachnidioidea  the  appendages  and 
branches  may  actually  fuse  histologically  and  may  produce  new  zooids  at  the  fusion 
sites  (Fig.  1 1). 

Tanganella  appendiculata  sp.  nov. 

Holotype  material:  Collection  of  D.  Jebram,  1978-10-10-2. 

Paratype  material:  U.  S.  National  Museum  of  Natural  History,  Smithsonian 
Institution  (Washington,  D.  C.),  Cat.  No.  36,  USNM  No.  292473;  Bryozoan  Col- 
lection of  the  Allan  Hancock  Foundation,  Univ.  of  Southern  California  at  Los 
Angeles,  No.  186.1;  British  Museum  (Natural  History),  London;  personal  collec- 
tions of  authors. 

Name:  The  cystids  usually  have  appendages  at  restricted  sites. 


VICTORELLIDAE  FROM  N.  AMERICA  177 

Synonyms:  part  of  the  material  of  Victorella  pavida  sensu  Osburn 
(1944),  according  to  his  description  (nee.  Victorella  pavida  Kent,  1870,  sensu 
Braem,  1951). 

Type  locality:  Lagoon  Pond,  Martha's  Vineyard  Island,  Massachusetts, 
U.  S.  A. 

Description:  The  colony  of  Tanganella  appendiculata  is  composed  of  serially 
arranged  zooids  (Fig.  5).  Each  zooid  usually  produces  one  distal  and  two  lateral 
daughter  zooids  on  the  sides  of  the  basal  part  of  the  cystid.  Older  zooids  can  form 
adventive  zooids  by  producing  "high  buds"  on  the  anal  and  lateral  sides  of  the 
peristomial  tube  (Fig.  6).  In  addition  to  the  encrusting  colony  parts  (forma  en- 
crustans)  free,  nonencrusting  branches  of  zooid  series  often  may  be  formed  at  the 
borders  of  substrate  pieces  or  may  originate  from  "high  buds"  (forma  ascendens). 
The  colony  form  of  this  species  thus  resembles  that  of  most  other  victorellids. 

The  zooids  are  composed  externally  of  two  main  parts,  a  basal  proximal  part 
(usually  encrusting  the  substrate)  and  an  upright  peristomial  tube.  The  basal  part 
is  somewhat  broadened  at  its  distal  part  and  gradually  narrows  proximally. 

Encrusting  zooids  of  Tanganella  appendiculata  usually  have  typical  cystid  ap- 
pendages. One  or  two  emerge,  usually  latero-proximally  on  each  side,  at  the  sites 
from  which  the  lateral  daughter  zooids  originate  from  the  basal  part  of  the  cystid. 
One  or  two  other  pairs  of  cystid  appendages  may  be  formed  anterior  to  the  budding 
places  of  the  side-branches  (Fig.  7).  However,  the  cystid  appendages,  especially 
the  distal  pair,  are  sometimes  vestigial  or  absent.  The  appendages  may  be  so  minute 
that  they  are  detectable  only  if  the  zooids  are  growing  on  glass  slides  and  are 
adequately  illuminated.  These  appendages,  of  course,  can  be  overlooked  easily  in 
specimens  from  the  natural  habitat,  especially  if  they  are  encrusting  a  rugged 
substrate.  The  cystid  appendages  sometimes  branch  into  several  tips  and  even  into 
four  separate  appendages.  In  this  species,  the  cystid  appendages  always  originate 
from  the  sides  of  the  distal,  broader  portion  of  the  encrusting  part  of  the  cystid, 
never  from  the  narrow,  proximal  portion.  Non-encrusting  zooids  produce  no  cystid 
appendages  (or  only  vestigial  ones).  The  appendages  from  different  zooids  may 
touch  each  other  but  never  fuse  histologically. 

The  younger  peristomial  tubes  are  always  considerably  inclined  distally;  older 
ones  may  become  raised  almost  perpendicular  to  the  basal  part  of  the  cystid.  The 
peristomial  tubes  grow  upward  in  steps  by  each  case  of  polypide  replacement  in 
the  same  cystid  and  may  attain  a  length  of  approximately  9  mm  (polypide  re- 
tracted). The  diameter  of  the  peristomial  tube  averages  160  /mi. 

The  autozooids  of  Tanganella  appendiculata  always  have  8  tentacles.  Sexually 
mature  zooids  have  an  intertentacular  pore  through  which  the  ova  are  released. 
This  very  narrow  pore  is  discernible  in  living  animals  only  during  the  release  of 
the  egg  through  the  pore. 

The  ova  are  apparently  fertilized  during  their  passage  through  the  interten- 
tacular pore.  At  that  time  they  are  dumbbell-shaped  or  irregularly  shaped  but  not 
spindle-shaped.  The  released  ova  are  pressed  to  the  anal  neck  region,  where  they 
adhere  to  the  body  wall.  Later  they  are  invaginated  into  a  pocket  of  the  body  wall; 
the  embryos  remain  there  until  they  develop  into  larvae  (Fig.  6).  Up  to  six  embryos 
may  be  brooded  in  the  median  line  of  the  anal  neck  region  of  one  zooid.  The 
polypide  apparently  remains  active  throughout  the  period  of  ova  release  (several 
days)  but  later  may  be  resorbed.  The  larvae  slip  through  the  breaking  body  wall 
into  the  water  and  may  swim  for  several  hours  or  days  (even  more  than  10  days!) 
until  they  find  an  acceptable  place  for  settlement.  The  first  polypide  of  the  ances- 
trula  of  Tanganella  appendiculata  has  6  tentacles. 


178 


D.  JEBRAM  AND  B.  EVERITT 


TanganeUa   appendiculata 


FIGURES  5-7:  TanganeUa  appendiculata  sp.  nov.  (5)  part  of  the  holotype  colony  demonstrating 
the  budding  pattern  in  an  encrusting  colony;  (6)  sexually  mature  zooid  with  adventive  zooid  and  "high 
buds,"  hb,  in  lateral  view;  (7)  some  zooids  in  basal  view  demonstrating  the  budding  pattern  and  the 
cystid  appendages;  ibe,  internally  brooded  embryos;  ip,  site  of  the  intertentacular  porus;  for  other  ab- 
breviations see  Figs.  1-4;  scale  bars  represent  1  mm. 


Lasting  buds  are  usually  formed  in  larger  colonies.  The  cuticle  of  older  lasting 
buds  becomes  light  brown  by  thickening.  The  reserves  of  the  lasting  buds,  the  ova, 
and  the  embryos  are  white. 


VICTORELLIDAE  FROM  N.  AMERICA  179 

The  anatomy  of  the  gut  of  Tanganella  appendiculata  is  similar  to  that  of  T. 
mulleri.  The  cardiac  sphincter  is  very  close  to  the  central  stomach,  a  characteristic 
given  by  Braem  (1951)  for  the  genus.  No  gizzard  is  formed.  The  caecum  is  con- 
siderably longer  and  more  slender  than  in  Victorella  in  proportion  to  the  size  of 
the  polypide. 

The  accompanying  fauna  in  the  type  locality  included  Bowerbankia  gracilis 
Leidy,  Barentsia  benedeni  (Foettinger),  and  Bulbella  abscondita  (see  below). 

Differentiating  characteristics  of  similar  forms:  (1)  Tanganella  mulleri  (Krae- 
pelin)  sensu  Braem  (1951)  forms  7  tentacles  in  the  first  polypide  generation  of  the 
ancestrula.  Braem  (1951),  who  made  most  of  his  observations  of  colonies  growing 
on  natural  (non-glass)  substrates,  did  not  describe  cystid  appendages  in  T.  mulleri. 
These  structures  have  now  been  detected  under  culture  conditions  for  both  species. 
Under  certain  salinity  ranges  and  dietary  conditions,  T.  mulleri  may  form  com- 
paratively smaller  cystid  appendages  at  5  sites  of  a  cystid,  2  latero-proximally  and 
3  distally  from  the  budding  sites  of  the  side  branches  (Fig.  1 1).  The  appendages, 
however,  especially  the  distal  ones,  are  often  absent  or  vestigial.  In  the  main 
branches,  the  encrusting  basal  cystid  parts  are  at  least  30%  shorter  in  T.  mulleri 
than  in  T.  appendiculata  (under  the  same  growth  conditions),  but  this  length  may 
vary  greatly  in  both  species  with  external  conditions.  As  Braem  (1951)  explained, 
contrary  to  the  assumptions  of  various  other  authors,  "Paludicella  mullen"  re- 
spectively Victorella  pavida  forma  mulleri  in  the  sense  of  Ulrich  (1926)  is  not 
Tanganella  mulleri  but  Bulbella  abscondita.  Nevertheless,  Prenant  and  Bobin 
(1956)  ignored  Braem's  (1951 )  correction  and  erroneously  maintained  the  incorrect 
identification  and  synonymy  of  Ulrich  (1926).  (2)  Victorella  pseudoarachnidia 
differs  from  Tanganella  appendiculata  in  the  location  of  the  cardiac  sphincter 
farther  above  the  central  stomach,  in  the  formation  of  an  intertentacular  tube,  in 
not  brooding  embryos,  and  in  the  form  and  arrangement  of  cystid  appendages. 
(3)  The  species  of  the  superfamily  Arachnidioidea  show  the  same  differences  as 
with  Victorella  pseudoarachnidia  (Victorelloidea). 

Bulbella  abscondita 

Paratype  material:  U.  S.  National  Museum  of  Natural  History,  Smithsonian 
Institution  (Washington,  D.  C),  Cat.  No.  36,  USNM  No.  292474;  Bryozoan  Col- 
lection of  the  Allan  Hancock  Foundation,  Univ.  of  Southern  California  at  Los 
Angeles,  No.  187.1;  British  Museum  (Natural  History),  London;  personal  collection 
of  authors. 

Locality:  Lagoon  Pond,  Martha's  Vineyard  Island,  Massachusetts,  U.  S.  A. 

Description:  In  most  features  the  specimens  found  in  this  study  resemble  those 
described  by  Braem  (1951).  The  colony  is  composed  of  serially  arranged  zooids 
(Fig.  8).  Each  zooid  usually  produces  one  distal  and  two  lateral  daughter  zooids. 
Adventive  zooids  originating  from  high-buds  occur  rarely  on  older  zooids.  In  old 
colonies,  the  zooids  are  crowded  and  grow  irregularly.  In  addition  to  the  encrusting 
zooids,  the  colony  rarely  may  produce  free,  non-encrusting  branches.  The  latter 
zooids  occur  mainly  at  the  borders  of  the  substrate  but  also  may  arise  from  the 
flat  areas;  their  production  is  related  partly  to  the  diet. 

The  young  zooids  are  comprised  almost  entirely  of  the  basal  cystid  part,  which 
is  broader  distally  and  slender  proximally  (Fig.  9).  Although  the  polypide  bud 
starts  to  develop  as  a  median  epidermal  invagination  (the  usual  process  in  cten- 
ostomes),  the  aperture  of  the  young  encrusting  zooids  is  always  lateral.  Within  one 


180 


D.  JEBRAM  AND  B.  EVERITT 


BulbeHa  abscondita 

FIGURES  8-10:  Bulbella  abscondita  (8)  and  (9)  parts  of  the  holotype  colony  demonstrating  the 
budding  pattern  in  an  encrusting  colony  (arrows  indicate  the  asymmetrical  position  of  the  apertural 
papillae);  (10)  sexually  mature  zooid  in  lateral  view;  apt,  apertural  papillae;  can,  external  annulations 
caused  by  too  strong  brushing;  ebe,  externally  brooded  embryos;  pb,  polypide  buds;  for  other  abbreviations 
see  Figs.  1-4;  scale  bars  represent  1  mm. 


distally  arranged  zooid  series,  the  right  or  left  position  of  the  initial  apertures  may 
vacillate  irregularly,  a  kind  of  enantiomorphic  effect.  Only  a  small  apertural  papilla 
is  formed  in  young  zooids  primarily  with  the  first  polypide  generation.  The  re- 
placement of  the  polypides  in  the  same  cystid  causes  a  modest  elongation  of  the 
peristomial  tube,  which  becomes  shifted  to  the  median  line  in  older,  crowded  zooids. 


VICTORELLIDAE  FROM  N.  AMERICA 


181 


SCHEMATICAL  SYNOPSIS  OF  TYPES  OF  CYSTID  APPENDAGES 

SERIALLY   ARRANGED   FORMS:  VICTORELLOIDS   (IN    BASAL  VIEW): 


Bulbella 
abscondita 


Tanganella 
appendiculata 


Tanganella 
miilleri 


Victorella 

pseudo- 

arachnidia 


STOLONATE   FORM,  VESICULARIOID: 


Buskia   nitens 


ARACHNIDIOID   FORM: 


Nolella    blakei 


FIGURE  1 1:  Schematical  synopsis  of  types  of  cystid  appendages  (in  basal  view).  In  the  victorellid 
species,  the  upper  zooid  demonstrates  the  ground  plan  of  the  arrangement  of  the  appendages,  while  the 
lower  zooid  shows  an  example  of  a  more  or  less  common  arrangement.  The  sketch  of  Buskia  nitens  is 
an  abstraction  from  various  observations  and  published  figures.  The  sketch  of  Nolella  blakei  is  redrawn 
from  Rogick  (1949,  fig.  5)  but  reversed  in  an  assumed  basal  view.  (All  examples  are  drawn  at  different 
scales.) 


The  peristomial  tubes  may  attain  a  length  of  approximately  3-4  mm  and  a  diameter 
of  approximately  260  nm. 

Encrusting  zooids  of  Bulbella  abscondita  often  form  cystid  appendages  on  the 
sides  of  the  broader,  distal  part  of  the  basal  region  of  the  cystid  but  never  on  the 


182  D.  JEBRAM  AND  B.  EVERITT 

narrower  proximal  part.  Of  four  potential  sites  from  which  an  appendage  may 
emerge,  0-4  may  actually  produce  one  (Figs.  8,  9,  11);  the  number  produced 
depends  partly  on  diet  conditions.  These  distinct  sites  for  the  cystid  appendages 
are  proximal  and  distal  to  the  budding  sites  of  the  side-branches.  The  cystid  ap- 
pendages of  different  zooids  may  touch  each  other  but  do  not  fuse  histologically. 

The  autozooids  of  Bulbella  abscondita  have  8  tentacles.  Sexually  mature  zooids 
have  an  intertentacular  tube  with  a  narrow  outlet  through  which  the  ova  are  released 
(Fig.  10).  The  ova  are  affixed  to  the  ano-median  line  (sometimes  to  the  ano-lateral 
side)  on  the  polypide  neck  region.  There  the  embryos  (up  to  6  per  zooid)  become 
larvae  but  are  not  invaginated  into  pockets  of  the  body  wall.  The  polypide  of  the 
mature  zooid  remains  active  during  the  ova-releasing  period.  The  color  of  the 
reserves  in  the  ova,  the  embryos,  and  the  young  larvae  varies  with  diet  and  ranges 
from  light  yellow  to  almost  white.  The  developed  larvae  are  released  and  may  swim 
for  a  few  hours  or  days  until  they  settle.  The  first  polypide  generation  of  the 
ancestrula  has  7  tentacles  (as  in  the  typical  form,  Braem,  1951). 

Some  dormant  buds,  which  were  hidden  under  older  zooids  and  were  growing 
on  glass  slides,  contained  very  light  yellowish  yolk  and  small  polypide  buds.  The 
dormant  buds  found  in  this  strain,  however,  have  usually  and  essentially  the  shape 
of  incompletely  developed  zooids  but  rarely  of  typical  lasting  buds  found  in  other 
ctenostomes. 

Braem  ( 1951 )  stated  that  Bulbella  abscondita  usually  has  a  gizzard  with  teeth 
but  that  the  dentation  may  range  from  fully  developed  dentation  to  complete  ab- 
sence of  teeth.  In  the  North  American  specimens,  a  muscular  proventriculus  is 
present,  but  a  true  gizzard  has  not  yet  been  observed.  The  cardiac  sphincter  is  far 
above  the  central  stomach  and  is  more  easily  discernible  in  living  animals  during 
typical  peristaltic  movements  of  the  gut.  The  caecum  is  comparatively  short  and 
stout.  (For  further  details  see  Braem,  1951.) 

Although  there  are  some  physiological  differences  between  the  North  American 
strain  and  the  Ryck  (Germany)  strain,  the  distinction  is  not  yet  sufficient  to  establish 
a  separate  species.  We  do  not  know  whether  the  North  American  form  can  penetrate 
rotten  wood  as  can  the  European  form.  The  German  form  of  Bulbella  is  being 
parallel-cultured  in  the  laboratory  at  Braunschweig. 

Differentiating  characteristics  of  similar  forms:  ( 1 )  The  species  of  the  super- 
family  Arachnidioidea  have  the  same  differences  as  with  Victorella  pseudoar- 
achnidia.  (2)  Buskia  nit  ens  has  true  stolons  limited  by  septa.  This  species  also 
prefers  higher  salinity  (down  to  polyhaline)  but  never  tolerates  oligohaline  con- 
ditions as  does  Bulbella. 

EXPERIMENTAL  BIOLOGY 

The  discrimination  of  species  in  the  Victorellidae  requires  living  and  sexually 
mature  animals  (Braem,  1951;  Jebram,  1969,  1976).  Two  main  factors  controlling 
growth  and  attainment  of  sexual  maturity  in  brackish-water  bryozoans  are  nutrition 
and  temperature  (Jebram,  1973a,  1975).  In  North  American  victorellids  discussed 
here,  these  factors,  especially  food,  were  investigated  by  various  qualitative  tests. 

Based  on  prior  studies  of  brackish-water  bryozoans  (Jebram,  1975,  1977b),  diet 
composition  for  the  new  bryozoan  strains  was  established  by  experience  (Jebram, 
1980b).  The  food  mixture  J5b  (Jebram,  1980b)  was  prepared  especially  for  the 
cultivation  of  Bulbella  abscondita  but  is  suitable  also  for  other  bryozoans.  Bulbella 
becomes  sexually  mature  with  this  diet.  Under  laboratory  conditions,  ova  and  em- 
bryos seemed  to  attain  the  typical  light  yellow  color  only  by  addition  of  those  food 


VICTORELLIDAE  FROM  N.  AMERICA  183 

species  containing  considerable  amounts  of  carotenoids,  e.g.,  haptophyceans,  chry- 
sophyceans,  bacillariophyceans.  Some  of  the  light  yellow  larvae  produced  with  this 
diet  metamorphosed  successfully  to  ancestrulae  and  initiated  the  formation  of  a 
new  colony  generation.  A  diet  mixture  containing  too  much  Cryptomonas  species 
caused  an  earlier  shifting  of  the  apertural  papilla  from  the  cystid  side  toward  the 
median  line  and  an  earlier  and  more  pronounced  elongation  into  a  peristomial  tube. 
The  latter  finding  agrees  with  the  observations  made  on  Bowerbankia  species  and 
Buskia  nitens  (Jebram,  1973a,  1973b). 

Although  Tanganella  mulleri,  T.  appendiculata,  and  Victorella  pseudoarach- 
nidia  thrived  and  matured  sexually  with  the  food  mix  J5b,  these  species  grew  much 
better  with  a  diet  including  Oxyrrhis  marina  (food  mix  J5h).  This  phagotrophic 
dinoflagellate  is  a  very  good  food  also  for  many  other  bryozoan  species  (Jebram, 
1969,  1975,  1980a,b).  Surprisingly,  Oxyrrhis,  presumably  due  to  its  taste,  is  very 
sparingly  accepted  by  Bulbella  abscondita.  Therefore,  Oxyrrhis  should  not  be  fed 
to  Bulbella  but  may  well  be  used  for  other  victorellids.  Diets  containing  O.  marina, 
however,  require  a  renewal  at  least  each  second  day  because  the  phagotrophic 
species  soon  alters  drastically  the  composition  of  the  diet  preparation.  Additionally, 
overaged  cultures  of  Oxyrrhis  may  have  toxic  effects  on  the  bryozoans  (Jebram, 
1975).  If  Oxyrrhis  is  used  as  a  mono-food  for  a  prolonged  period,  unusual  growth 
forms  may  result.  Further  details  concerning  general  problems  of  the  nu- 
trition of  bryozoans  have  been  discussed  earlier  (Jebram,  1977a,b,  1979,  1980a,b). 

Even  under  the  same  external  conditions  (e.g.,  food,  salinity,  temperature),  all 
four  victorellids  cultivated  in  the  Braunschweig  laboratory  (Victorella  pseudo- 
arachnidia,  Tanganella  appendiculata,  T.  mulleri,  Bulbella  abscondita}  had  dif- 
ferent growth  rates  and  formed  different  colony  habits  (qualitative  observations). 
Although  the  different  colony  habits  can  be  observed  easily  when  the  specimens 
are  side  by  side,  these  differences  can  be  described  less  easily.  This  difficulty  was 
perhaps  one  of  the  reasons  for  the  confusion  in  the  taxonomy  of  this  group  of 
bryozoans  in  the  past.  Victorella  pseudoarachnidia  exhibited  the  fastest  growth 
rate  and  formed  larger  bushes  of  the  forma  ascendens.  The  zoaria  of  Tanganella 
appendiculata  colonized  the  substrate  more  quickly  (by  greater  elogation  of  the 
narrow  proximal  cystid  part)  than  those  of  T.  mulleri,  but  the  latter  attained  sexual 
maturity  sooner.  In  Victorella  and  Tanganella  the  growth  rate  and  the  formation 
of  adventive  zooids  by  high  buds  were  considerably  greater,  and  sexual  maturity 
occurred  earlier  with  the  food  mixture  J5h  (with  O.  marina)  than  with  J5b.  Bulbella 
abscondita  showed  the  slowest  growth  rate. 

The  formation  of  cystid  appendages  was  apparently  more  or  less  influenced  by 
unknown  dietary  components  in  all  the  victorellids  cultivated.  Additionally,  in  lower 
salinity  ranges  (5-8%o),  Tanganella  mulleri  formed  no  (or  only  vestigial)  append- 
ages, but  in  T.  appendiculata  the  appendages  only  became  small  (or  were  sometimes 
absent).  With  greater  salinity  (about  15%o)  the  formation  of  the  appendages  in- 
creased in  both  species  of  Tanganella.  In  Victorella  pseudoarachnidia,  however, 
the  growth  of  cystid  appendages  seemed  unaffected  by  variation  of  salinity  within 
ecologically  acceptable  ranges. 

Tanganella  mulleri,  T.  appendiculata,  and  Bulbella  abscondita  inhabit  areas 
in  which  the  water  temperature  seldom  reaches  and  rarely  exceeds  20°C.  Accord- 
ingly, these  species  attained  sexual  maturity  in  the  laboratory  at  temperatures  of 
19°C  or  lower.  On  the  other  hand,  the  Salton  Sea  (California),  from  which  Vic- 
torella pseudoarachnidia  was  collected,  is  in  a  warm  semi-desert  area  (water  tem- 
perature 26°C  at  0900  on  9  Oct.  1977).  Therefore  this  species  must  be  adapted 
to  higher  temperature  ranges.  Accordingly,  V.  pseudoarachnidia  seemed  to  require 


184  D.  JEBRAM  AND  B.  EVERITT 

a  temperature  above  20°C  for  sexual  maturation  in  the  laboratory  but  grew  well 
asexually  at  lower  temperatures. 

DISCUSSION 
Cystid  appendages  in  ctenostome  taxonomy 

One  reason  for  seeking  ctenostomatous  bryozoans  in  Lagoon  Pond  on  Martha's 
Vineyard  Island  (Mass.,  U.  S.  A.)  in  September  of  1977  was  Rogick's  (1949) 
report  of  Nolella  blakei,  which  she  thought  lived  in  that  pond.  Although  her 
specimens  of  N.  blakei  have  not  been  found  again,  her  description  is  undoubtedly 
that  of  an  arachnidioid  species.  Instead  of  the  expected  species,  we  were  surprised 
to  find  an  obvious  victorelloid,  a  species  of  Tanganella. 

Rogick  (1949)  noted  that  she  collected  benthos  from  Lagoon  Pond  but  that 
Nolella  blakei  was  not  seen  initially  in  that  material.  She  stored  the  material  in 
watch  glasses  in  large  aquaria  which  were  supplied  with  running  sea  water  piped 
from  the  nearby  bay.  After  nine  days  she  discovered  a  ctenostome  in  those  glasses 
and  described  N.  blakei.  It  now  appears  that  N.  blakei  does  not  inhabit  the  brackish 
Lagoon  Pond  but  that  it  is  a  marine  species  (like  most  other  species  of  Nolella} 
and  that  Rogick's  colony  originated  from  larvae  in  the  seawater  piped  from  the 
bay.  Rogick  did  not  mention  any  species  of  the  Victorelloidea  in  her  Lagoon  Pond 
material. 

The  cystid  appendages  of  Tanganella  appendiculata  were  not  observed  in  our 
first  specimens  from  Lagoon  Pond,  in  which  they  grew  crowded  with  Bowerbankia 
gracilis  on  natural  substrates.  When  the  Tanganella  material  was  cultured  on  glass 
slides  in  the  laboratory  at  Braunschweig,  the  cystid  appendages  were  detected. 
Another  unexpected  discovery  was  that  the  European  Tanganella  mulleri  can  also 
produce  cystid  appendages  under  certain  conditions;  Braem  (1951)  did  not  describe 
such  appendages  in  T.  mulleri. 

These  observations  generated  two  basic  questions.  First,  are  there  two  separate 
species  of  serially  arranged  ctenostomes  in  the  Salton  Sea  (California)  as  reported 
by  Soule  (1957)7  The  senior  author  examined  Soule's  specimens  and,  after  addi- 
tional studies  on  living  animals,  concluded  that  Soule's  "Nolella  blakei"  is  identical 
with  his  Victorella  "pavida".  Soule's  specimens  of  "Nolella"  were  actually  those 
zooids  of  Victorella  which  were  growing  on  glass  bottles.  The  cystid  appendages 
on  glass  can  be  seen  more  easily  than  on  other  substrates  and  also  can  be  removed 
more  readily.  Soule's  Victorella  "pavida"  was  mainly  material  from  other  types 
of  substrate,  and  the  cystid  appendages  were  probably  lost  or  damaged  during 
removal  of  the  zooids.  One  of  Soule's  drawings  (1957,  Fig.  c)  shows  zooids  of  K 
"pavida"  with  appendages,  but  the  morphology  of  the  appendages  was  not  suffi- 
ciently analyzed.  Such  analysis  requires  proper  procurement  and  preparation  of 
specimens.  The  following  features  common  to  all  known  serially  arranged  cteno- 
stomes from  the  Salton  Sea  indicate  that  they  all  belong  to  one  species,  a  Victorella: 
constant  number  of  tentacles  (8);  intertentacular  tube;  absence  of  brooding  of 
embryos;  anatomy  of  the  gut;  difference  in  arrangement  and  growth  potential  of 
cystid  appendages  as  compared  to  arachnidioid  species  (see  below). 

The  second  basic  question  is  whether  the  presence  of  cystid  appendages  in  the 
true  victorellids  means  that  there  are  no  principal  differences  between  the  Victo- 
rellidae  and  the  Arachnidiidae.  Such  a  separation  has  been  doubted  by  some  earlier 
authors.  A  close  examination  of  anatomical  details  reveals  the  general  differences 
between  victorelloid  appendages  and  arachnidioid  appendages.  In  victorellids  the 


VICTORELLIDAE  FROM  N.  AMERICA  185 

appendages  originate  at  distinct  and  more  or  less  limited  sites  on  the  cystids,  whether 
or  not  the  potential  appendages  actually  develop,  and  the  appendages  are  never 
produced  on  the  narrow  and  most  proximal  cystid  part.  In  the  Arachnidioidea, 
however,  appendages  may  be  formed  irregularly  at  various  sites  of  the  cystid  bor- 
ders, including  the  narrow  proximal  cystid  part.  Appendages  of  different  zooids 
may  (but  not  necessarily)  fuse  histologically  and  often  produce  a  new  zooid  at  those 
points  of  fusion  in  arachnidioid  species.  This  histological  fusion  does  not  occur  in 
the  victorelloid  (and  some  stolonate)  species  (see  Fig.  1 1). 

In  addition  to  observations  of  collected  specimens,  studies  on  living  colonies  of 
Cryptoarachnidium  argilla  have  revealed  the  absolutely  different  growth  potential 
of  the  arachnidioid  cystid  appendages.  Banta  ( 1967)  originally  described  this  species 
as  Victorella  argilla.  Re-examination  of  paratype  specimens  showed  that  this  spe- 
cies is  undoubtedly  not  a  victorelloid  but  an  arachnidioid  species,  and  the  new 
genus  Cryptoarachnidium  was  established  (Jebram,  1973b).  Specimens  from  Ma- 
rina del  Rey  (California,  U.  S.  A.)  have  been  cultivated  since  October  of  1977  in 
Braunschweig. 

Cystid  appendages  apparently  have  developed  independently  in  various  phy- 
logenetic  lines  in  the  Ctenostomata.  Appendages  are  typical  for  most  species  of  the 
Arachnidioidea  but  are  formed  also  in  several  species  of  the  Walkerioidea,  e.g., 
Aeverrillia  setigera  (Hincks),  and  the  Vesicularioidea,  e.g.,  species  of  Bowerbankia, 
Buskia,  and  Cryptopolyzoon.  This  study  has  revealed  that  cystid  appendages  are 
common  also  in  the  Victorelloidea.  In  the  latter  superfamily,  cystid  appendages 
simply  have  been  overlooked  in  some  of  the  species  in  the  past.  The  important 
characteristic  for  the  placement  of  a  species  in  a  ctenostome  superfamily,  then,  is 
not  the  presence  or  absence  of  cystid  appendages  but  the  details  of  their  arrange- 
ment and  growth  potential. 

Cultivation  experiments  as  a  taxonomic  technique 

Rogick  agreed  with  Soule  (1957)  that  some  of  the  Salton  Sea  specimens  were 
Nolella  blakei.  The  confusion  of  those  workers  resulted  mainly  from  the  fact  that 
the  available  animals  were  already  preserved  and  were  sexually  immature.  Volu- 
minous ecological  surveys  and  monographs  (e.g.,  Schiitz,  1963;  Carrada  and  Sacchi, 
1964)  are  less  valuable  if  they  are  based  partially  or  mainly  on  incorrect  identi- 
fication of  some  of  the  predominant  primary  consumers  (bryozoans)  in  brackish 
habitats. 

In  microbiology  and  botany,  experimental  work  as  an  aid  for  identification  of 
species  has  a  long  tradition.  In  zoology,  however,  cultivation  techniques  for  tax- 
onomic purposes  have  been  used  for  relatively  few  taxa,  e.g.,  protozoans,  some 
polychaetes  and  platyhelminths,  certain  parasitic  or  pathogenic  species,  and,  of 
course,  for  various  genetics  studies.  Most  taxonomists  and  most  workers  in  faunistics 
and  synecology  traditionally  study  preserved  specimens.  Although  Braem  (1951) 
demonstrated  the  usefulness  of  living  animals  for  some  more  delicate  taxonomic 
problems,  this  approach  has  been  virtually  neglected  in  other  earlier  bryozoan 
studies.  The  results  described  above  reveal  that  the  taxonomy  in  the  Victorellidae, 
including  reports  from  Asia  and  Africa,  can  be  sufficiently  determined  only  on  the 
basis  of  living  animals  and  with  parallel-culture  methods  under  defined  conditions. 
There  should  be  no  doubt  that  taxonomy  in  various  groups  of  the  Bryozoa  (and 
other  taxa)  requires  experimental  work  and  detailed  studies  on  living  animals.  In 
some  cases,  future  taxonomic  investigation  must  include  not  only  morphological 
features  already  present  but  also  the  growth  potential  of  the  zooids. 


186  D.  JEBRAM  AND  B.  EVERITT 

ACKNOWLEDGMENTS 

Both  authors  thank  very  much  Dr.  M.  B.  Abbott,  Dr.  F.  P.  Bowles,  and  Dr. 
J.  D.  Ebert  for  their  support  during  our  stay(s)  in  the  Marine  Biological  Laboratory 
in  Woods  Hole,  Massachusetts  in  September,  1977,  and  in  August,  1978.  Jebram 
is  indebted  to  Dr.  R.  R.  Given  (Santa  Catalina  Marine  Science  Center,  California), 
Dr.  R.  Feldmeth  (Claremont  Colleges,  Los  Angeles),  Dr.  D.  F.  Soule,  Dr.  J.  D. 
Soule,  Dr.  R.  L.  Zimmer,  and  Dr.  Oguri  (University  of  Southern  California,  Los 
Angeles),  and  Linda  and  Michael  Boss  (Brawley,  California)  for  their  hospitality 
and  help  during  his  stay  in  California  in  1977.  The  experimental  work  on  the 
nutrition  of  the  saltwater  bryozoa  is  supported  by  the  Deutsche  Forschungsge- 
meinschaft  (grants  no.  Je  62/9-11). 

LITERATURE  CITED 

BANTA,  W.  C.  1967.  A  new  species  of  Viclorella  from  Southern  California  (Bryozoa,  Ctenostomata). 
Proc.  U.  S.  Nat.  Museum  Washington  122(3593):  1-18. 

BRAEM,  F.  1911.  Beitrage  zur  Kenntnis  der  Fauna  Turkistans,  7.  Bryozoen  und  deren  Parasiten.  Trav. 
Soc.  Nat.  St.  Petersbourg  42(2):  1-35. 

BRAEM,  F.  1951.  Uber  Victorella  und  einige  ihrer  nachsten  Verwandten,  sowie  iiber  die  Bryozoenfauna 
des  Ryck  bei  Greifswald.  Zoologica  (Stuttgart)  37(102):  1-59. 

BRATTSTROM,  H.  1954.  Notes  on  Victorella  pavida  Kent.  Acta  Univ.  Lundensis  (2)  50(9):  1-29. 

CARRADA,  C.  C.,  AND  C.  F.  SACCHI.  1964.  Recherches  ecologiques  sur  le  bryozoaire  ctenostome  Vic- 
torella pavida  (Kent).  Vie  el  Millieu  15(2):  389-428. 

EVERITT,  B.  1975.  Fresh-water  Ectoprocta:  Distribution  and  ecology  of  five  species  in  southeastern 
Louisiana.  Trans.  Amer.  Microscop.  Soc.  94:  130-134. 

HYMAN,  L.  H.  1959.  Part  V,  Smaller  coelomate  groups,  Pp.  273-515  in  The  Invertebrates.  McGraw- 
Hill,  New  York  and  London. 

JEBRAM,  D.  1969.  Bryozoen  als  Holschadlinge  im  Brackwasser.  Kieler  Meeresforsch.  25:  224-231. 

JEBRAM,  D.  1973a.  Preliminary  observations  on  the  influences  of  food  and  other  factors  on  the  growth 
of  bryozoa  with  the  description  of  a  new  apparatus  for  cultivation  of  sessile  plankton  feeders. 
Kieler  Meeresforsch.  29:  50-57. 

JEBRAM,  D.  1973b.  Stolonen-Entwicklung  und  Systematik  bei  den  bryozoa  Ctenostomata.  Z.  Zoo/.  Syst. 

Evolutions-forschung  11(1):  1-48. 
JEBRAM,  D.  1975.  Effects  of  different  foods  on  Conopeum  seurati  (Canu)  (Bryozoa,  Cheilostomata) 

and  Bowerbankia  gracilis  Leidy  (Bryozoa,  Ctenostomata).  Docum.  Lab.  Geol.  Fac.  Sci.  Lyon 

H.  S.  3(Fasc.  1):  97-108. 
JEBRAM,  D.  1976.  Bryozoa  Ctenostomata  in  the  lakes  near  Naples.  Pubbl.  Staz.  Zoo/.  Napoli  40:  73- 

82. 
JEBRAM,  D.  1977a.  Experimental  techniques  and  culture  methods.  Pp.  273-306,  chapter  10  in:  Biology 

of  Bryozoans.  R.  M.  Woollacott  and  R.  L.  Zimmer,  Eds.  Academic  Press,  New  York. 
JEBRAM,  D.  1977b.  Culture  media  and  diets  for  bryozoa.  Pp.  77-92.  in:  CRC  Handbook  Series  in 

Nutrition  and  Food.  M.  Rechcigl,  Jr.  (Editor-in-Chief),  Sec.  G,  Vol.  II,  CRC-Press,  Cleveland, 

Ohio. 
JEBRAM,  D.  1979.  Interrelations  of  nutrition,  food  uptake,  and  growth  in  bryozoans.  Pp.  121-140  in: 

Advances  in  Bryozoology.  G.  P.  Larwood  and  M.  B.  Abbott,  Eds.  Academic  Press,  London 

and  New  York. 
JEBRAM.  D.  1980a.  Prospection  for  a  sufficient  nutrition  for  the  cosmopolitic  marine  bryozoan,  Electro 

pilosa  (Linnaeus).  Zoo/.  Jb.  Syst.  Okol.  Geograph.  Tiere  107:  368-390. 
JEBRAM,  D.  1980b.  Laboratory  diets  and  qualitative  nutritional  requirements  for  bryozoans.  Zoo/.  Anz. 

(Jena)  205:  333-344. 
KENT,  S.  1870.  On  a  new  polyzoon,  Victorella  pavida,  from  the  Victoria  Docks.  Quart.  J.  Microscop. 

Sci.  (n.  s.)  10:  34-39. 
KRAEPELIN,  K.   1887.  Die  deutschen  Susswasser-Bryozoen.  Eine  Monographic.  I.  Anatomisch-syste- 

matischer  Teil.  Abhandlg.  Naturw.  Hamburg  10(9):  5-168. 
OSBURN,  R.  C.  1944.  A  survey  of  the  bryozoa  of  Chesapeake  Bay.  Chesapeake  Biol.  Lab.  Publication 

no.  63:  1-58. 


VICTORELLIDAE  FROM  N.  AMERICA  187 

PRENANT,  M.,  AND  G.  BOBIN.  1956.  Bryozoaires,  Premie  Partie  Entoproctes,  Phylactolemes,  Cteno- 

stomes.  "Faune  de  France"  60:  1-398.  Librairie  de  la  Faculte  des  Sciences,  Paris. 
POIRRIER,  M.  A.,  AND  M.  M.  MULING.  1977.  Effects  of  environmental  factors  on  the  distribution  and 

morphology  of  Viclorella  pavida  (Ectoprocta)  in  Lake  Pontchartrain,  Louisiana,  and  vicinity. 

Chesapeake  Sciences  18(4):  347-352. 
ROGICK,  M.  D.  1949.  Studies  on  marine  bryozoa.  IV.  Nolella  blakei  n.  sp.  Biol.  Bull.  (Woods  Hole, 

Mass.)  97(2):  158-168. 
SACCHI,  C.  F.,  AND  CARRADA,  C.  C.  1962.  Ciclo  morfologico ed  euriecia  in  Viclorella  Pavida  (Bryozoa, 

Ctenostomata)  al  lago  Fusaro  (Napoli).  Natura  53:  43-56. 
SCHUTZ,  L.  1963.  Okologische  Untersuchungen  iiber  die  Benthosfauna  im  Nordostseekanal.  I.  Auto- 

kologie  der  Arten.  Int.  Revue  gesamt.  Hydrobiologie  48(3):  361-418. 
SOULE,  J.  D.  1957.  Two  species  of  Bryozoa  Ctenostomata  from  the  Salton  Sea.  Bull.  South.  Calif. 

Acad.  Sci.  56(1):  21-30. 
ULRICH,  W.  1926.  Uber  das  Vorkommen  der  Victorella  pavida  Kent  und  einiger  anderer  Bryozoen  im 

Brackwasser  des  Rostocker  Hafens.  Z.  Morph.  Okol.  Tiere  5:  559-579. 


Reference:  Biol.  Bull.  163:  188-196.  (August,  1982) 


THE  RELEASE  OF  THE  PEDAL  DISK  IN  AN  UNDESCRIBED  SPECIES 
OF  TEALIA  (ANTHOZOA:  ACTINIARIA) 

I.  D.  LAWN1*  AND  D.  M.  ROSS2** 

*Bamfield  Marine  Station,  Bamfield,  British  Columbia,  Canada  VOR  I  BO;  and  2 Department  of 
Zoology,  University  of  Alberta.  Edmonton,  Alberta,  Canada  T6G  2E9 

ABSTRACT 

Specimens  presumed  to  belong  to  an  undescribed  species  of  Tealia  were  col- 
lected subtidally  in  the  northeast  Pacific.  In  contact  with  the  asteroids  Dermasterias 
imbricata  and  Patiria  miniata,  these  animals  expanded  their  oral  disks,  constricted 
their  columns,  and  detached  their  pedal  disks.  Other  asteroids  had  no  such  effect. 
Of  five  other  species  of  Tealia,  only  T.  piscivora  showed  similar  behavior  and  only 
to  D.  imbricata.  Electrophysiological  records  showed:  1 )  that  D.  imbricata  evokes 
pulses  in  a  slow  conduction  system  (McFarlane's  SSI);  and  2)  that  a  train  of 
electrical  stimuli  also  causes  SSI  pulses  and  brings  about  the  release.  It  is  concluded 
that  SSI  pulses  trigger  the  releasing  behavior  of  Tealia  sp.  as  they  do  the  release 
and  swimming  behavior  of  Stomphia  spp.  A  review  of  pedal  disk  release  in  the 
actinians  shows  that  it  occurs  only  in  certain  genera  and  species  in  several  families 
not  closely  related.  Although  the  circumstances  and  functions  of  the  release  where 
known  are  not  the  same  in  different  species,  the  neurophysiological  mechanisms 
employed  are  strikingly  similar.  Also  discussed  are  the  active  role  of  the  pedal  disk 
in  special  behavior  patterns  and  a  possible  function  of  the  release  in  the  escape  of 
Tealia  sp.  from  a  predator. 

INTRODUCTION 

Sea  anemones  with  adhesive  pedal  disks  generally  remain  firmly  fixed  to  the 
substratum.  If  they  change  positions  at  all  they  do  so  by  extremely  slow  sliding 
steps  across  the  surface.  However,  a  few  anemones  detach  their  pedal  disks  rapidly 
in  response  to  specific  stimuli.  Calliactis parasitica  (Couch)  does  so  in  its  symbiotic 
interactions  with  hermit  crabs  (Ross,  1967,  1974),  and  Stomphia  coccinea  (Miiller) 
and  S.  didemon  Siebert  (Siebert,  1973)  also  do  so  when  they  encounter  certain 
asteroids  and  aeolids,  moving  away  afterwards  by  repeated  flexions  of  the  body 
(Yentsch  and  Pierce,  1955;  Sund,  1958;  Robson,  1961).  These  activities  have  pro- 
vided opportunities  for  studying  the  behavioral  physiology  of  actinians  in  general 
and  have  contributed  to  the  discovery  of  the  conduction  systems  that  control  be- 
havior in  these  animals  (McFarlane,  1969a,b;  Lawn,  1976). 

The  present  study  began  with  the  observation  that  specimens  believed  to  belong 
to  Tealia  crassicornis  released  their  pedal  disks  when  they  came  into  contact  with 
the  leatherstar,  Dermasterias  imbricata.  Attempts  to  confirm  this  gave  variable 
results,  a  difficulty  that  was  only  removed  later  when  Sebens  and  Laakso  (1977) 
showed  that  some  anemones  previously  assigned  to  T.  crassicornis  ranked  as  a 
separate  species  which  they  named  T.  piscivora.  When  we  tested  both  species 
separately  we  found  that  specimens  of  T.  crassicornis  never  released  their  pedal 

Received  2  November  1981;  accepted  18  May  1982. 

*  Present  address:  Heron  Island  Marine  Station,  Gladstone,  Queensland  4680,  Australia. 

**  Author  to  whom  reprint  requests  should  be  addressed. 

188 


PEDAL  DISK  RELEASE  IN  TEALIA  189 

disks  in  response  to  D.  imbricata  whereas  specimens  of  T.  piscivora  usually  did  so. 
About  the  same  time  specimens  of  an  undescribed  north  Pacific  Tealia  were  col- 
lected which  gave  an  even  more  striking  releasing  response  to  D.  imbricata.  The 
behavioral  physiology  of  this  animal  is  the  subject  of  this  paper.  Pending  a  full 
systematic  description  we  shall  name  it  Tealia  sp.  A  laboratory  and  subtidal  study 
is  now  in  progress  on  the  behavior  and  general  ecology  of  T.  piscivora.  This  will 
be  the  subject  of  a  separate  paper. 

MATERIALS  AND  METHODS 

Tealia  sp.  was  collected  in  Barkley  Sound,  British  Columbia,  at  depths  of  75- 
110m.  Exact  locations  and  depths  are  not  known  because  all  8  specimens  obtained 
during  1978-80  came  up  on  separate  occasions  in  a  fisherman's  net.  Another 
specimen  was  found  in  the  display  tank  at  the  Friday  Harbor  Laboratory  in  1981, 
but  the  site  and  time  of  its  collection  are  not  known.  We  present  here  a  few  features 
of  the  8  animals  in  our  collections:  diameter  of  the  tentacular  crown,  5-20  cm,  and 
of  the  pedal  disk,  3- 1 4  cm;  height  of  column  4-20  cm;  color  of  column,  translucent, 
grading  through  pale  mauve  to  pink  below  the  margin;  tiny  beadlike  verrucae  on 
the  column  in  irregular  horizontal  rows.  The  animal  was  judged  to  be  a  species  of 
Tealia  from  the  decamerous  arrangement  of  the  innermost  ring  of  tentacles,  the 
presence  of  a  fosse,  the  long  stout  tentacles,  and  the  presence  of  verrucae  resembling 
those  of  some  other  species  of  Tealia.  It  did  not  correspond  to  any  known  species 
of  Tealia  as  described  by  Stephenson  (1935),  Carlgren  (1949),  and  Hand  (1955). 
With  so  few  animals  available,  priority  was  given  to  behavioral  and  physiological 
work  on  the  living  anemones  before  preserving  specimens  for  identification. 

The  animals  were  kept  in  aquaria  at  the  Bamfield  Marine  Station  and  were  fed 
about  once  per  week  on  pieces  of  fish  or  mollusks.  D.  imbricata  and  other  asteroids 
were  presented  to  individual  anemones  and  their  responses  noted  and  timed.  Typical 
responses  were  recorded  in  still  and  motion  pictures  for  further  study. 

Electrophysiological  techniques  followed  the  standard  procedure  developed  for 
sea  anemones  (McFarlane,  1969a,b;  Lawn,  1976,  1980).  A  polyethylene  suction 
electrode  was  attached  to  a  tentacle  for  recording  purposes,  and  a  similar 
stimulating  electrode  was  attached  to  the  column. 

RESULTS 
Behavioral  observations 

The  behavior  of  Tealia  sp.  and  five  described  species  of  Tealia  (T.  coriacea, 
T.  crassicornis,  T.  lofotensis,  T.  columbiana,  T.  piscivora}  was  first  studied  in  10 
presentation  trials  with  Dermasterias  imbricata.  The  sea  stars  were  brought  into 
contact  with  firmly  attached  anemones  and  kept  in  contact  for  3  min  or  until  the 
anemone  detached  its  pedal  disk.  Tealia  sp.  released  its  pedal  disk  in  9  and  T. 
piscivora  in  8  of  the  10  trials.  None  of  the  other  species  ever  responded  in  this 
way.  The  tentacles  of  species  that  did  not  release  normally  clung  strongly  to  the 
sea  star  as  to  food,  whereas  Tealia  sp.  and  T.  piscivora  remained  in  contact  without 
clinging. 

Specimens  of  14  other  asteroids  were  available  at  Bamfield  for  trials  with  Tealia 
sp.  similar  to  those  described  above  with  Dermasterias.  Only  one  of  these  species, 
Patiria  miniata,  caused  frequent  release  of  the  pedal  disk,  7  times  in  20  trials.  Two 
other  species  caused  the  pedal  disk  to  release  occasionally:  Solaster  stimpsoni  once 
in  20  trials;  Crossaster  papposus  once  in  14  trials.  The  following  11  species,  each 


190  I.  D.  LAWN  AND  D.  M.  ROSS 

tested  20  times,  never  caused  the  pedal  disk  to  release:  Evasterias  troscheli,  Hen- 
ricia  leviscula,  Hippasteria  spinosa,  Leptasterias  hexactis,  Mediaster  aequalis, 
Orthasterias  koehleri,  Pisaster  brevispinus,  Pisaster  ochraceus,  Pteraster  tesse- 
latus,  Pycnopodia  helianthoides,  and  Solaster  dawsoni. 

Release  times  provided  further  evidence  that  Dermasterias  (mean  time  33  sec 
in  14  releases)  was  considerably  more  effective  than  Patiria  (mean  time  78  sec  in 
7  releases).  The  single  releases  to  Crossaster  papposus  and  Solaster  stimpsoni  took 
place  at  130  and  180  sec,  respectively.  These  results  suggest  that  the  release  is 
triggered  by  substances  which  are  present  and  deliverable  in  amounts  that  can 
cause  release  frequently  in  only  two  of  the  asteroids  tested.  Possibly  these  substances 
occur  in  other  asteroids  also  but  only  at  levels  that  are  usually  below  the  threshold 
that  causes  the  release  of  the  pedal  disk. 

Unlike  the  responses  of  Stomphia  spp.  to  Dermasterias,  etc.,  the  release  of  the 
pedal  disk  in  Tealia  sp.  is  not  accompanied  or  followed  by  asymmetrical  flexions 
("swimming")  or  other  repetitive  activity.  Resettlement  often  followed  quickly, 
within  2-5  min,  if  the  anemone  remained  upright.  When  the  anemone  fell  over  or 
was  carried  away  by  a  current,  resettlement  did  not  begin  until  the  pedal  disk  came 
against  a  surface  to  which  it  could  adhere. 

One  of  the  8  specimens  of  Tealia  sp.  was  much  larger  than  the  others  (pedal 
disk  diameter  14  cm).  This  animal  failed  to  release  in  response  to  Dermasterias 
on  a  number  of  occasions;  in  fact  most  of  the  trials  which  failed  to  cause  release 
occurred  with  this  animal.  We  found  that  the  small  and  medium-sized  specimens 
of  Tealia  sp.  in  our  small  collection  gave  more  consistent  and  more  rapid  responses, 
and  they  were  used  more  frequently  in  our  tests.  If  a  larger  supply  of  animals  could 
become  available  it  would  be  interesting  to  see  if  a  relationship  exists  between  the 
size  of  the  anemone  and  the  frequency  and  speed  of  the  release. 

Figures  1-4  show  a  typical  release  of  Tealia  sp.  when  its  tentacles  were  touched 
by  D.  imbricata.  On  a  moderately  extended  specimen  with  the  sea  star  touching 
the  tentacles  (Fig.  1 )  the  diameter  of  the  tentacular  crown  increased,  the  oral  disk 
became  convex,  and  the  column  shortened  dramatically  to  about  one-quarter  of  its 
original  length  (Fig.  2).  These  changes  took  place  by  slow,  smooth  movements, 
almost  imperceptible  as  they  happened  but  transforming  the  animal  completely  in 
less  than  half  a  minute. 

The  shortening  of  the  column  was  due  in  part  to  a  constriction  of  the  margin 
of  the  pedal  disk  to  form  a  tightly  contracted  ring.  Consequently  the  pedal  disk 
no  longer  adhered  and  the  anemone  became  detached  and  took  on  the  shape  of  an 
inverted,  almost  medusoid,  cone  with  the  tentacular  crown  flared  out  and  the  pedal 
disk  deeply  concave  (Fig.  3).  Later,  as  the  anemone  began  to  resettle,  about  3  min 
from  the  beginning,  the  column  extended,  and  the  constricted  pedal  disk  began  to 
adhere  and  extend  outwards  to  bring  the  animal  back  to  its  normal  position  and 
appearance  (Fig.  4). 

Figures  5-8  show  another  example.  This  animal  had  a  short  column  at  the 
beginning  (Fig.  5).  About  15  sec  after  Dermasterias  was  brought  in,  the  pedal  disk 
lifted  following  the  narrowing  of  the  base  (Fig.  6)  and  the  flaring  out  of  the  crown 
and  oral  disk.  Side  views  show  the  detached  and  almost  flat  pedal  disk  with  a 
pattern  of  concentric  grooves  and  at  the  center  a  slight  elevation  with  a  small 
central  pit  (Fig.  7).  Later  the  base  narrowed  and  resembled  a  terminal  knob  with 
the  upper  margin  and  crown  still  flared  out  and  completely  inactive,  giving  the 
animal  the  shape  of  a  flower  vase  (Fig.  8).  Then  the  pedal  disk  slowly  assumed  its 
normal  dimensions  and  settlement  proceeded  as  the  disk  spread  across  the  stone. 
The  entire  response  took  less  than  10  min. 


PEDAL  DISK  RELEASE  IN  TEALIA 


191 


FIGURES  1-8.  Tealia  sp.  Two  examples  of  pedal  disk  release  and  subsequent  changes  in  shape  in 
response  to  contact  with  Dermasterias  imbricata.  Full  description  in  text.  Time  elapsed  between  Figures 
1  and  4  approx.  2.5  min  and  between  Figures  5  and  8  approx.  3.0  min. 

There  were  minor  variations  around  these  patterns  in  the  responses  of  our 
animals.  Examples  of  some  of  the  shapes  assumed  at  various  times  are  shown  in 
Figures  9-16.  It  often  happened  also  that  an  animal  seemingly  about  to  resettle 


192 


I.  D.  LAWN  AND  D.  M.  ROSS 


FIGURES  9-16.  1'calia  sp.  Characteristic  postures  after  release  of  pedal  disk  in  response  to  Der- 
masterias.  Note  release  and  shortening  of  column  (9,  10),  swelling  of  pedal  disk  with  upturned  basal 
margin  (11,  12,  13),  extension  of  column  with  intermittent  peristaltic  waves  (12,  14,  15,  16). 

would  fail  to  do  so  and  would  assume  strange  shapes  and  postures  with  strong 
peristaltic  waves  passing  orally,  before  finally  settling  down. 

The  nudibranch,  Aeolidia  papillosa,  which  can  cause  Stomphia  to  detach  and 
swim,  induced  detachment  in  Tealia  sp.  in  three  out  of  1 1  trials.  The  release  times 


PEDAL  DISK  RELEASE  IN  TEALIA  193 


I 
DERMASTERIAS 


PDR 

Ll  I          ^ 

IJV^M,  ,«»,,,  ,,.„.,,«..,,.. .  J»%«'^<.^.VW^,.^W.^*MI>/\><^^  "y*»* 


Is 

FIGURE  17.  Electrical  activity  recorded  from  tentacles  of  Tealia  sp.  during  and  following  2.8  sec 
contact  between  Dermasterias  and  tentacles  of  anemone.  Note  7  typical  SSI  pulses.  Arrow  at  PDR 
marks  time  of  release  of  pedal  disk.  (Continuous  trace;  duration  approx.  17  sec). 

were  long,  75,  80  and  240  sec.  The  responses  differed  from  the  response  to  the 
asteroids.  Instead  of  spreading  the  oral  disk  outward,  the  anemone  closed  up  and 
then  released  its  pedal  disk.  It  is  interesting  to  note  that  Hippasteria  spinosa,  an 
asteroid  that  is  highly  successful  in  causing  Stomphia  to  detach  and  swim,  had  no 
obvious  effect  on  Tealia  sp. 

Electrophysiological  data 

McFarlane  ( 1969a)  and  Lawn  (1976)  have  described  three  types  of  pulses  that 
have  been  recorded  from  sea  anemones  using  standard  electrophysiological  tech- 
niques. These  pulses  are  attributed  to  three  different  conduction  systems  that  have 
become  known  by  their  initials:  1 )  NN  (through-conducting  nerve  net  located  in 
the  endoderm);  2)  SSI  (slow  system  1  located  in  the  ectoderm);  3)  SS2  (slow 
system  2  located  in  the  endoderm).  Each  system  has  been  linked  to  specific  behavior 
patterns,  e.g.  NN  with  retraction  and  closure.  SSI  pulses  coincide  with  the  release 
of  the  pedal  disk  in  two  cases:  in  Calliactis  parasitica  SS 1  pulses  accompany  the 
slow  release  of  the  pedal  disk  in  transfers  to  shells  from  other  surfaces  (McFarlane 
1969b,  1973);  in  Stomphia  coccinea  SSI  pulses  precede  the  release  of  the  pedal 
disk  in  encounters  with  Dermasterias  (Lawn,  1976).  In  a  European  Tealia,  T. 
felina  var.  lofotensis  (nomenclature  of  Stephenson,  1935),  SSI  pulses  triggered  the 
prefeeding  response  that  occurs  when  food  substances  come  into  contact  with  the 
column  (McFarlane,  1970;  Lawn,  1975).  Thus  there  was  reason  to  believe  that 
SSI  pulses  would  be  recorded  in  Tealia  sp.  and  special  interest  attached  to  finding 
out  whether  SSI  pulses  were  associated  also  in  this  animal  with  the  release  of  the 
pedal  disk. 

Figure  17  shows  a  typical  record  of  the  electrical  activity  in  Tealia  sp.  when 
Dermasterias  imbricata  was  brought  up  to  the  tentacles  for  a  few  seconds.  Two 
SSI  pulses  of  characteristic  appearance  occurred  about  2  sec  after  contact.  Two 
more  pulses  followed  at  1  sec  intervals  and  three  more  followed  with  the  intervals 
between  pulses  becoming  longer  until  the  anemone  released  its  pedal  disk,  about 
14  sec  after  the  sea  star  made  contact.  This  strongly  suggests  that  the  detachment 
of  the  pedal  disk  in  Tealia  sp.  is  triggered  by  a  series  of  SSI  pulses. 


194  I.  D.  LAWN  AND  D.  M.  ROSS 

The  record  shown  in  Figure  17  was  preceded  by  a  trace  lasting  several  minutes 
from  which  SSI  pulses  were  conspicuously  absent.  Similar  records  were  obtained, 
with  pretrial  controls,  from  three  other  specimens  of  Tealia  sp.  Further  trials  were 
carried  out  which  gave  similar  records  when  the  anemones  had  resettled.  The 
relationship  between  SSI  pulses  and  the  release  of  the  pedal  disk  as  seen  in  Figure 
17  is  not  a  statistical  event;  in  our  experience  the  two  invariably  occur  together. 

The  SSI  pulses  recorded  before  the  detachment  of  Tealia  sp.  are  almost  iden- 
tical with  those  that  precede  the  detachment  of  the  swimming  anemone  Stomphia 
(Lawn,  1976).  The  time  that  elapses  between  the  application  of  the  stimulus,  the 
sea  star,  and  the  beginning  of  the  response,  is  of  the  same  order,  usually  a  few 
seconds.  The  number  of  SSI  pulses  preceding  detachment  is  also  of  the  same  order 
in  the  two  cases,  usually  from  6  to  12. 

Examining  records  from  a  number  of  interactions  between  D.  imbricata  and 
Tealia  sp.  shows  a  good  deal  of  variation  in  the  number  and  the  timing  of  pulses 
that  trigger  the  release  of  the  pedal  disk  on  different  occasions.  A  recurrent  feature 
of  the  records  was  a  tendency  for  two  or  three  pulses  to  occur  close  together  soon 
after  contact  was  established  between  the  two  animals. 

Pateria  miniata,  as  reported  earlier,  sometimes  evoked  the  releasing  behavior, 
and  it  also  set  up  trains  of  SSI  pulses.  Of  10  trials,  four  resulted  in  detachment. 
The  pulses  in  these  records  were  similar  to  those  in  the  interactions  with  Dermas- 
terias,  though  generally  the  firing  rate  was  lower,  and  in  cases  where  release  did 
not  occur  the  pulses  ceased  after  an  initial  two  or  three.  Maintaining  the  activity 
after  these  first  few  pulses  seems  to  be  important  to  the  triggering  function. 

Electrical  stimulation  of  the  SSI  in  Tealia  sp.  caused  detachment  of  the  pedal 
disk  and  inflation  of  the  oral  disk.  It  was  not  possible  to  cut  ectodermal  flaps 
successfully  in  this  anemone  (McFarlane,  1969b),  and  this  meant  that  the  SSI 
could  not  be  stimulated  separately  from  the  nerve  net.  Results  showed  that  the 
effective  frequencies  of  stimulation  fell  in  the  range  of  one  shock  every  3  sec  to  one 
shock  every  10  sec.  The  minimum  number  of  shocks  required  to  produce  a  response 
varied  from  four  to  eight  depending  on  frequency.  This  corresponds  closely  to  the 
situation  previously  encountered  in  Stomphia  (Lawn,  1976). 

DISCUSSION 

These  results  give  rise  to  discussion  on  three  topics:  1 )  comparative  aspects;  2) 
the  activity  and  mobility  of  the  pedal  disk;  3)  the  adaptational  significance  of  the 
releasing  behavior  in  Tealia  sp. 

Comparative  aspects 

Detachment  in  response  to  specific  stimuli  seems  to  be  a  general  adaptation  in 
a  few  genera  in  certain  families.  Boloceroides,  in  the  family  Boloceroididae,  is  a 
lightly  attached  Indo-Pacific  anemone  that  releases  quickly  and  swims  actively  in 
response  to  a  nudibranch  predator  (Lawn  and  Ross,  1982).  Stomphia  spp.  in  the 
large  family  Actinostolidae,  few  of  whose  species  have  been  observed  alive,  release 
quickly  and  "swim"  in  response  to  certain  sea  stars  and  nudibranchs  (Robson, 
1966;  Ross,  1974).  Calliactis  spp.,  and  other  symbiotic  actinians  in  the  family 
Hormathiidae  that  live  on  crustaceans  and  gastropods,  release  their  pedal  disks 
slowly  in  response  to  shells  or  to  the  manipulations  of  certain  hermit  and  spider 
crabs  (Ross,  1974). 

Tealia  belongs  to  the  Actiniidae,  most  of  which  are  very  firmly  attached  and 
difficult  to  dislodge,  e.g.  Actinia  spp.  This  description  applies  to  the  familiar  north- 
east Pacific  species,  T.  coriacea  and  T.  crassicornis.  The  fact  that  Tealia  sp.  and 


PEDAL  DISK  RELEASE  IN  TEALIA  195 

T.  piscivora  can  release  quickly  in  interactions  with  certain  animals  in  their  en- 
vironments, whereas  four  other  species  of  Tealia  cannot  do  so,  shows  that  behavioral 
attributes  often  differ  within  taxa  of  generic  or  higher  rank;  within  a  genus  or 
family  these  attributes  may  be  restricted  to  particular  species  only.  We  see  in  this 
another  example  of  the  versatility  of  neuromuscular  mechanisms  in  the  actinians, 
often  without  any  external  signs  of  such  special  adaptations. 

It  is  instructive  to  compare  the  behavioral  physiology  of  Tealia  sp.  and  Stomphia 
spp.  in  releasing  the  pedal  disk.  The  SSI  conduction  system  is  activated  in  both 
cases  by  the  same  stimulus,  Dermasterias.  The  SSI  pulses  trigger  the  release  in 
similar  ways;  the  number  of  pulses  required,  their  frequency,  and  the  latency  of 
the  response  are  of  the  same  order  of  magnitude  in  both  animals.  However,  the 
behavioral  events  that  accompany  and  follow  the  release  differ.  In  Tealia  sp.  the 
column  shortens  and  the  oral  disk  and  tentacles  flare  out  into  an  immense  corona 
and  contain  most  of  the  coelentric  fluid.  After  this  transformation,  the  anemone 
slowly  returns  to  normal  and  resettles.  The  entire  behavior  proceeds  without  any 
movements  except  slow  symmetrical  changes  of  shape.  Moreover,  there  is  no  evi- 
dence of  the  post-release  or  post-swimming  torpor  of  Stomphia  spp.  Once  Tealia 
sp.  releases,  the  tentacles  become  extremely  adhesive,  unlike  those  of  Stomphia 
which  are  non-adherent  at  this  time.  Whereas  in  Tealia  sp.,  the  column  is  greatly 
shortened  during  the  release,  in  Stomphia  it  is  greatly  extended,  and  the  release 
is  accompanied  and  followed  by  swimming  flexions  for  2-3  min.  Thus,  almost 
identical  triggering  systems  are  used  in  the  two  cases  to  achieve  different  ends. 

The  pedal  disk 

These  results  reemphasize  the  sensory  and  motor  activity  taking  place  in  the 
pedal  disk  in  these  special  behavior  patterns.  Earlier  examples  were:  the  demon- 
stration by  Davenport  et  al.  (1961)  that  the  clinging  of  the  tentacles  of  Calliactis 
on  shells  depended  on  information  as  to  whether  the  pedal  disk  was  on  a  shell  or 
not;  the  description  of  the  release  and  the  resettlement  of  Calliactis  and  Stomphia 
showing  the  pedal  disk  to  be  an  area  in  which  many  activities  take  place,  e.g.,  the 
symmetrical  constriction  bringing  about  release  in  Calliactis  (Ross  and  Sutton, 
1961);  the  swelling  of  the  pedal  disk  to  make  contact  with  surfaces  for  settling  in 
Stomphia  and  Paracalliactis  (Ross,  1974);  the  description  of  the  asymmetrical 
locomotory  movements  in  Metridium  (Batham  and  Pantin,  1950).  The  behavior 
of  Tealia  sp.  described  above  provides  another  example  of  the  activity  of  the  pedal 
disk  in  actinian  behavior,  especially  in  anemones  that  abandon  their  sedentary 
habits  from  time  to  time. 

Adaptational  significance 

The  adaptational  significance  of  the  detachment  behavior  of  Tealia  sp.  is  far 
from  clear.  By  analogy  it  looks  like  an  escape  response  but  there  is  no  evidence 
that  asteroids  prey  on  Tealia  sp.  If  it  is  an  escape  reaction,  the  anemone  may 
employ  it  to  escape  from  some  other  predator  not  yet  discovered.  Once  detached, 
the  expanded  Tealia  sp.  is  virtually  weightless  so  that  any  current  would  carry  it 
away  and  remove  it  from  a  potential  predator.  However,  questions  about  the  ad- 
aptational significance  of  the  releasing  behavior  in  Tealia  sp.  can  only  be  answered 
with  data  from  subtidal  observations.  Such  studies  are  now  in  progress  with  T. 
piscivora.  Unfortunately,  Tealia  sp.  has  been  collected  infrequently,  its  normal 
habitat  is  unknown,  and  it  has  not  yet  been  located  by  divers,  so  we  have  no 
immediate  prospect  of  observing  it  in  nature. 


196  I.  D.  LAWN  AND  D.  M.  ROSS 

ACKNOWLEDGMENTS 

The  release  of  Tealia  piscivora  in  response  to  an  asteroid  was  first  observed  by 
Dr.  W.  Kokke,  at  the  time  our  colleague  in  an  investigation  on  the  chemistry  of 
Dermasterias.  We  acknowledge  his  contribution  with  pleasure  and  thank  him  for 
his  continuing  interest  in  this  work.  We  also  thank  Miss  Sandy  Walde,  a  student 
at  the  University  of  Calgary,  for  assistance  in  the  collection  of  behavioral  and 
electrophysiological  data.  The  support  of  Operating  Grant  No.  A- 1445  to  D.M.R. 
from  the  Natural  Sciences  and  Engineering  Research  Council  of  Canada  is  grate- 
fully acknowledged. 

LITERATURE  CITED 

BATHAM,  E.  J.,  AND  C.  F.  A.  PANTIN.  1950.  Phases  of  activity  in  the  sea-anemone,  Metridium  senile 

(L.),  and  their  relation  to  external  stimuli.  J.  Exp.  Biol.  27:  337-399. 
CARLGREN,  O.  1949.  A  survey  of  the  Ptychodactiaria,  Corallimorpharia  and  Actiniaria.  Kgl.  Sv.  Ve- 

tenskapsakad.,  Handl.  1:  1-129. 
DAVENPORT,  D.,  D.  M.  Ross,  AND  L.  SUTTON.  1961.  The  remote  control  of  nematocyst  discharge  in 

the  attachment  of  Calliactis  parasitica  to  shells  of  hermit  crabs.  Vie  Milieu  12:  197-209. 
HAND,  C.  1955.  The  sea  anemones  of  Central  California.  Part  II.  The  endomyarian  and  mesomyarian 

anemones.  Wasmann  J.  Biol.  13:  37-99. 
LAWN,  I.  D.  1975.  An  electrophysiological  analysis  of  chemoreception  in  the  sea  anemone  Tealia  felina. 

J.  Exp.  Biol.  63:  525-536. 
LAWN,  I.  D.  1976.  Swimming  in  the  sea  anemone  Stomphia  coccinea  triggered  by  a  slow  conduction 

system.  Nature  262:  708-709. 
LAWN,  I.  D.   1980.  A  transmesogloeal  conduction  system  in  the  swimming  sea  anemone  Stomphia 

coccinea.  J.  Exp.  Biol.  83:  45-52. 
LAWN,  I.  D.,  AND  D.  M.  Ross.   1982.  The  behavioural  physiology  of  the  swimming  sea  anemone 

Boloceroides  mcmurrichi.  Proc.  Roy.  Soc.  Lond.  Ser.  B.  (in  press). 
McFARLANE,  I.  D.  (1969a).  Two  slow  conduction  systems  in  the  sea  anemone  Calliactis  parasitica. 

J.  Exp.  Biol.  51:  377-385. 

MCFARLANE,  I.  D.  1969b.  Co-ordination  of  pedal-disk  detachment  in  the  sea  anemone  Calliactis  par- 
asitica. J.  Exp.  Biol.  51:  387-396. 
McFARLANE,  I.  D.  1970.  Control  of  preparatory  feeding  behaviour  in  the  sea  anemone  Tealia  felina. 

J.  Exp.  Biol.  53:  211-220. 
MCFARLANE,  I.  D.  1973.  Multiple  conduction  systems  and  the  behaviour  of  sea  anemones.  Publ.  Seto 

Mar.  Biol.  Lab.  20:  513-523. 
ROBSON,  E.  1961.  Some  observations  on  the  swimming  behaviour  of  the  anemone  Stomphia  coccinea. 

J.  Exp.  Biol.  38:  343-363. 
ROBSON,  E.  1966.  Swimming  in  Actiniaria.  Pp.  333-360  in  W.  J.  Rees,  Ed.,  The  Cnidaria  and  their 

evolution.  Academic  Press,  New  York. 

Ross,  D.  M.  1967.  Behavioural  and  ecological  relationships  between  sea  anemones  and  other  inverte- 
brates. Oceanogr.  Mar.  Biol.  Annu.  Rev.  5:  291-316. 
Ross,  D.  M.  1974.  Behavior  patterns  in  associations  and  interactions  with  other  animals.  Pp.  281-312 

in  L.  Muscatine  and  H.  M.  Lenhoff,  Eds.,  Coelenterate  biology.  Reviews  and  new  perspectives. 

Academic  Press,  New  York. 
Ross,  D.  M.,  AND  L.  SUTTON.  1961.  The  response  of  the  sea  anemone  Calliactis  parasitica  to  shells 

of  the  hermit  crab  Pagurus  bernhardus.  Proc.  Roy.  Soc.  Lond.  Ser.  B  155:  266-281. 
SEBENS,  K.  P.,  AND  G.  LAAKSO.  1977.  The  genus  Tealia  (Anthozoa:  Actiniaria)  in  the  waters  of  the 

San  Juan  Archipelago  and  the  Olympic  Peninsula.  Wasmann  J.  Biol.  35:  152-168. 
SIEBERT,  A.  E.  1973.  A  description  of  the  sea  anemone  Stomphia  didemon  sp.  nov.  and  its  development. 

Pac.  Sci.  27:  363-376. 

STEPHENSON,  T.  A.  1935.  The  British  sea  anemones.  Vol.  II.  Ray  Society,  London.  426  pp. 
SUND,  P.  N.  1958.  A  study  of  the  muscular  anatomy  and  swimming  behaviour  of  the  sea  anemone, 

Stomphia  coccinea.  Quart.  J.  Microsc.  Sci.  99:  401-420. 
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1231-1233. 


Reference:  Biol.  Bull.  163:  197-210.  (August,  1982) 


PUMPING  RATES  AND  PARTICLE  RETENTION  EFFICIENCIES  OF 
THE  LARVAL  LAMPREY,  AN  UNUSUAL  SUSPENSION  FEEDER 

JON  MALLATT 
Department  of  Zoology,  Washington  State  University,  Pullman,  WA  99164 

ABSTRACT 

The  suspension  feeding  larvae  of  lampreys  (ammocoetes)  inhabit  fine-grained 
sediments  where  participate  organic  matter  is  concentrated,  but  whose  low  per- 
meability limits  the  rate  at  which  ammocoetes  can  pump  water  (flow  rate).  This 
study  determined:  1 )  flow  rates  through  the  pharynges  of  ammocoetes,  both  within 
and  out  of  the  sediment,  and  2)  the  ability  of  ammocoetes  to  filter  particles  from 
suspension  (retention  efficiency)  over  a  wide  range  of  algal  cell  concentrations 
(Chlorella  pyrenoidosa,  1-75  mg/1). 

For  most  suspension  feeders,  flow  rate  and  retention  efficiency  must  be  measured 
indirectly  (clearance  method).  Direct  measurement  was  possible  here,  as  ammo- 
coetes remain  apparently  undisturbed  in  glass  tubes  that  allow  the  separation  of 
inhalent  from  exhalent  ventilatory  currents.  Problems  arise  in  attempting  to  use 
clearance  methods  to  determine  flow  rates  in  burrowed  suspension  feeders,  and 
these  problems  are  discussed. 

Ammocoete  flow  rates  are  exceptionally  low  compared  to  the  rates  of  other 
suspension  feeders,  but  retention  efficiency  was  consistently  high,  even  at  the  highest 
algal  concentrations  employed  (x  =  82%).  While  most  suspension  feeders  rapidly 
process  dilute  suspensions,  ammocoetes  meet  nutrient  needs  by  slowly  processing 
concentrated  suspensions. 

INTRODUCTION 

Lampreys  spend  most  of  their  life  cycle  as  suspension  feeding  larvae  (ammo- 
coetes), living  within  the  sediment  of  stream  beds  (Hardisty  and  Potter,  1971; 
Potter,  1980).  Ammocoetes  occupy  burrows  that  are  either  open  at  one  end  (mouth) 
or  are  fully  closed  off  from  the  overlying  water.  Suspended  food  particles  are 
obtained  from  the  water  just  above  the  substrate  surface,  and  also  from  pore  water 
within  the  sediment  (Moore  and  Mallatt,  1980).  Feeding  involves  trapping  small 
particulate  detritus  and  unicellular  algae  on  mucus  within  the  pharynx  (Mallatt, 
1979,  1981).  Water  is  propelled  by  rhythmic  muscular  contractions  of  the 
pharyngeal  wall,  and  by  a  pair  of  muscular  flaps,  the  velum,  at  the  anterior  end 
of  the  pharynx  (Rovainen  and  Schieber,  1975).  Observations  in  this  laboratory 
indicate  ammocoetes  extrude  their  exhalent  water  into  the  substrate  around  the 
burrow.  Since  the  sediments  occupied  are  fine  sands  and  muds  (see  Fig.  1,  and 
Malmqvist,  1980)  of  low  permeability,  ammocoetes  must  pump  water  against  re- 
sistance. The  thick,  particle-trapping  mucus,  which  fills  most  of  the  pharynx  (Mal- 
latt, 1981),  also  is  likely  to  impede  water  flow. 

Received  16  November  1981;  accepted  18  May  1982. 

Abbreviations:  F,  rate  of  water  flow  through  pharynx;  F',  clearance  rate;  RE,  retention  efficiency; 
W,  wet  weight  of  larvae. 


197 


198 


JON  MALLATT 


SEDIMENT  GRAIN  SIZE 


PARTICLE   DIAM.,  urn 


FIGURE  1.  Size-frequency  distributions  of  particles  comprising  several  sediments  in  which  am- 
mocoetes  will  burrow  and  feed.  That  labelled  'a'  is  a  diatomaceous  earth,  'b'  and  V  are  commercially 
obtained  70-mesh  silica  sands,  and  'd'  is  a  sand  from  an  ammocoete  habitat  (Petromyzon  marinus,  Pere 
Marquette  River,  Michigan).  Sand  'c'  was  used  throughout  this  study.  The  numbers  of  grains  measured 
exceeded  175  in  all  cases.  As  a  measure  of  permeabilities,  the  times  taken  to  drain  10  cm  of  water 
through  10  cm  sediment  columns  are:  a:  21  min;  b:  6.5  min;  c:  2  min;  d:  2.75  min. 


A  search  of  the  literature  on  suspension  feeding  animals  (see  especially  J0r- 
gensen,  1966,  and  Wallace  and  Merritt,  1980)  revealed  no  other  instance  in  which 
water  is  characteristically  propelled  into  a  fine-grained  substrate.  Most  suspension 
feeders  are  either  pelagic  (crustacean  zooplankton:  Jorgensen,  1975;  rotifers:  Stark- 
weather, 1980;  frog  tadpoles:  Scale  et  al.,  1982),  or  if  benthic,  are  epifaunal  (bi- 
valves: Winter,  1973;  ascidian  tunicates:  Randl0v  and  Riisgard,  1979).  The  infaunal 
suspension  feeders  that  have  been  studied  either  inhabit  coarse  permeable  sediment 
(amphioxus:  Azariah,  1969;  Webb,  1975)  or  have  full  access  to  the  overlying  water. 
Such  access  is  achieved  through  U-shaped  burrows  (mayfly  and  midge  larvae), 
siphons  with  inhalent  and  exhalent  openings  (infaunal  bivalves),  or  protruding  the 
filter  into  the  overlying  water  (many  polychaetes).  In  pumping  against  resistance, 
ammocoetes  are  unusual  among  suspension  feeders.  In  fact,  the  existence  of  factors 
threatening  to  limit  the  rate  at  which  ammocoetes  can  pump  water  seems  to  clash 
with  the  basic  tenet  of  suspension  feeding  that  large  quantities  of  water  must  be 
processed  rapidly  (J0rgensen,  1975).  How  does  such  a  suspension  feeder  survive? 

Another  unusual  feature  of  ammocoete  ecology  is  that  the  habitat  contains 
comparatively  high  concentrations  of  suspended  food  particles.  Due  to  natural  pro- 
cesses of  particle  settling  and  resuspension,  suspensions  are  expected  to  be  more 
concentrated  at  the  floor  of  a  natural  body  of  water  than  in  the  water  column  above 
(Hardisty  and  Potter,  1971).  Supporting  this,  Moore  (Moore  and  Potter,  1976, 
Fig.  Ib;  Moore  and  Mallatt,  1980,  Fig.  1)  measured  higher  levels  of  suspended 
organic  solids  at  the  substrate  in  ammocoete  habitats  (1-40  mg/1)  than  typically 
are  present  in  open  waters,  where  many  other  suspension  feeders  are  found  (below 
1  mg/1;  J0rgensen,  1975).  The  nature  of  their  habitat  suggests  ammocoetes  can 
efficiently  process  concentrated  suspensions,  and  this  merits  experimental  investi- 
gation. 

With  the  special  ecological  features  in  mind,  this  work  determines  the  rates  at 
which  ammocoetes  pump  water  (flow  rate)  when  in  and  out  of  sediment,  and  the 


LARVAL  LAMPREY  FEEDING  199 

efficiency  with  which  they  remove  food  particles  (retention  efficiency)  from  sus- 
pensions of  different  concentrations. 

Larval  lampreys  are  ideal  experimental  animals  for  this  type  of  study.  For  most 
other  suspension  feeders,  flow  rate  and  retention  efficiency  must  be  estimated  in- 
directly, through  monitoring  the  rates  at  which  they  clear  particles  from  the  water 
(clearance  rates:  see  J0rgensen,  1975,  for  discussion).  To  utilize  such  a  method, 
however,  one  must  assume  at  some  point  that  retention  efficiency  is  100%,  an 
assumption  that  is  untestable  for  most  animals.  For  ammocoetes,  flow  rate  and 
retention  efficiency  can  be  measured  directly,  as  the  larvae  will  feed  in  tubes,  which 
allow  separation  of  inhalent  and  exhalent  currents. 

This  study  provides  some  kinds  of  data  seldom  obtained  for  suspension  feeders. 
Flow  rates  are  measured  in  the  absence  of  food  particles,  not  possible  with  indirect 
methods.  Also,  this  is  one  of  the  first  studies  in  which  retention  efficiency  is  in- 
vestigated as  a  function  of  particle  concentration  (also  see  Kurtak,  1978).  In  most 
past  studies  that  have  employed  direct  techniques  on  suspension  feeders  (Fiala- 
Medioni,  1978;  Randl0v  and  Riisgard,  1979),  retention  efficiency  was  related  only 
to  particle  size. 

Data  on  the  flow  rates  of  ammocoetes  in  glass  tubes  are  supplemented  by 
clearance  rate  data  from  burrowed  individuals.  Special  problems  arise  in  attempting 
to  use  indirect  methods  to  determine  the  flow  rates  of  burrowing  suspension  feeders 
in  situ,  and  these  are  documented  here. 

MATERIALS  AND  METHODS 

This  study  primarily  utilized  larval  Petromyzon  marinus,  which  were  obtained 
from  the  Muskegon  and  Pere  Marquette  rivers,  Michigan,  and  from  the  Hammond 
Bay  Biological  Station,  Millersburg,  Michigan.  A  few  Pacific  lamprey  ammocoetes 
(Lampetra  tridentatus}  were  used,  obtained  from  the  Potlatch  River  near  Bovill, 
Idaho.  (Ammocoetes  of  different  species  are  quite  similar,  morphologically  and 
physiologically:  Hardisty  and  Potter,  1971.)  Stock  animals  fed  on  yeast  and  grew 
normally,  averaging  a  10%  weight  increase  per  month  (Mallatt,  unpublished).  Ex- 
perimental animals  were  between  10.1  and  11.1  cm  long,  with  wet  weights  between 
1.3  and  2.0  g  (x  -  1.6).  All  experiments  were  performed  at  12°C. 

The  use  of  glass  tubes  to  measure  flow  rate  was  inspired  by  Rovainen  and 
Schieber  (1975).  Test  chambers  were  glass  pans  (Fig.  2)  divided  into  anterior  and 
posterior  compartments,  holding  150  and  1 100  ml  of  water,  respectively.  The  water 
was  dechlorinated  tap  water,  previously  filtered  through  a  0.45  jum  Millipore®  filter; 
water  was  continuously  aerated  in  both  compartments.  The  test  animal  occupied 
a  glass  tube,  which  pierced  the  partition.  P.  marinus  larvae  were  employed  whose 
pharynges  fit  snugly  but  without  constriction  into  the  tubes  (0.6  cm  internal  di- 
ameter). Flow  rates  were  monitored  in  dim  light.  Water  pumped  by  the  animal 
from  the  anterior  compartment  was  replaced  continuously,  and  the  flow  rate  was 
considered  to  be  the  replacement  rate  (ml/hr,  later  adjusted  for  animal  mass). 
Differences  in  water  height  between  anterior  and  posterior  chambers  were  kept  low 
(<0.5  cm).  It  was  determined,  through  removal  and  addition  of  known  amounts 
of  water,  that  the  mean  and  maximum  errors  in  the  measure  of  flow  rate  were 
±2  and  ±5  ml/hr,  respectively.  In  preliminary  tests,  dye  (Methyl  blue)  added  to 
the  posterior  compartment  did  not  color  water  in  the  anterior  compartment  over 
an  eight  hour  period  with  the  ammocoete  in  place,  so  flow  was  unidirectional  as 
expected. 


200 


JON  MALLATT 


tight   fit 


FIGURE  2.  Apparatus  employed  for  measuring:  1)  flow  rate  through  the  pharynx,  and  2)  particle 
retention  efficiency,  of  larval  P.  marinus.  Ammocoete  (a)  in  the  tube  (t)  pumps  water  from  the  anterior 
compartment  (A)  to  the  posterior  compartment  (P).  Inset  shows  tube  in  dorsal  view.  Other  symbols: 
b,  buret;  ba,  balloon  attachment  site;  e,  eye;  ebp,  external  branchiopores;  m,  mouth;  p,  pharynx. 


The  experimental  procedure  involved  monitoring  the  flow  rates  of  12  individual 
ammocoetes  for  periods  of  4  to  13  hours,  after  an  1 8  hour  period  of  adjustment 
to  the  apparatus.  The  reason  flows  were  monitored  over  time  was  to  determine 
whether  the  confinement  of  the  tubes  stressed  the  ammocoetes,  as  might  be  reflected 
in  a  cumulative  tendency  to  increase  or  decrease  flow  rate  (Cairns  et  al.,  1982). 

For  measuring  the  clearance  rates  of  buried  ammocoetes,  an  indirect  technique 
was  used,  similar  to  that  of  Malmqvist  and  Bronmark  (1982).  The  test  chambers 
were  five-liter  aquaria,  containing  four  liters  of  continuously  aerated,  dechlorinated 
tap  water,  one  liter  of  which  occupied  the  interstices  in  two  liters  of  a  silica  sand 
(Fig.  1,  sand  'c').  Two  test  aquaria  were  employed,  one  containing  P.  marinus,  and 
the  other,  L.  tridentatus  ammocoetes  (four  to  six  per  tank,  8-12  g).  Each  test  tank 
was  paired  with  a  control  tank  that  lacked  ammocoetes. 

This  test  of  clearance  rates  lasted  two  months,  with  trials  conducted  daily.  At 
the  onset  of  each  trial,  fresh  yeast  suspension  (Saccharomyces  cerevisiae,  Fleisch- 
mann's®  cakes)  was  added  to  the  water  above  the  sediment  of  test  and  control 
tanks,  in  amounts  that  varied  randomly  from  trial  to  trial,  to  yield  particle  con- 
centrations ranging  from  5  to  2700  mg  (dry)  per  liter.  Yeast  cell  concentrations 
in  the  water  above  the  sand  (C)  were  measured  visually  with  a  hemocytometer  at 
the  onset  (C,)  and  the  end  (C2)  of  the  four  to  eight  hour  duration  (T)  of  each 


LARVAL  LAMPREY  FEEDING 


201 


trial.  The  rates  at  which  burrowed  ammocoetes  cleared  particles  from  the  overlying 
water  (F',  ml/g/h)  were  calculated  according  to  the  equation: 

3000  ml  [(In  Cu  --  In  C2.t)  -  (In  CliC  -  In  C2.c)] 

T-W 


Clearance  Rate  =  F'  = 


where  3000  ml  is  the  volume  of  overlying  water,  W  is  the  wet  weight  of  the  larvae 
in  the  test  tank,  and  the  subscripts  t  and  c  denote  test  and  control  tanks,  respectively 
(Coughlan,  1969).  The  water  was  changed  and  the  sand  was  washed  after  each 
trial.  Trials  were  conducted  in  the  dark.  Variation  in  cell  settling  rates  in  control 
versus  test  tanks  led  to  the  occasional  calculation  of  negative  clearance  values; 
these  were  treated  as  zero  (or  as  one  ml/g/h  for  log-transformed  data). 

Another  experiment  was  performed  to  relate  flow  rates  measured  for  ammo- 
coetes in  tubes  to  the  clearance  rates  of  burrowed  individuals.  Here,  the  effect  of 
the  sand's  resistance  on  flow  rate  was  tested  directly.  Three  P.  marinus  larvae  that 
had  been  used  in  clearance  studies  were  placed  in  the  tube  devices.  A  flexible  plastic 
tube  was  then  fit  snugly  around  the  posterior  end  of  each  glass  tube,  and  the  rate 
at  which  the  ammocoete  propelled  water  through  the  plastic  tubing  was  monitored 
before  and  after  the  insertion  of  a  plug  of  sand.  The  latter  was  4  cm  long,  ap- 
proximating the  depth  at  which  many  buried  ammocoetes  resided  in  stock  tanks. 
The  plastic  tube  was  bent  slightly  to  assure  the  sand  entirely  filled  the  width  of  its 
lumen. 

Several  things  should  be  noted  about  the  construction  of  Table  I,  where  flow 
rates  are  compared  among  many  groups  of  suspension  feeders.  These  figures  can 


TABLE  I 


Flow  rate  in  different  suspension  feeders 


Animal 


Wet  weight 
(g) 


Flow  rate 
(ml/g/h) 


Flow  rate 

adjusted  to  1.6  g 

wet  body  mass" 


1.  Copepods 

a.  Calanus  helgolandicus 

b.  Calanus  pacificus 

2.  Lamellibranches 

a.  Various  bivalvesb 

(13  species) 

b.  Crassostrea  virginica 

c.  Pecten  irradians 

d.  Mvtilus  edulis 

e.  Various  bivalvesb  (3  species) 

f.  Dreissena  polymorpha 

3.  Cladocerans 

a.  Daphnia  pulex 

b.  Daphnia  magna 

4.  Rotifers 

a.  Keratella  cochlearis  (large) 

b.  Keratella  cochlearis  (small) 

c.  Conochilus  dossuarius 

d.  Kellicottia  bostoniensis 

5.  Sponges 

a.  Sycon  coronatum 

b.  Halichondria  panica 


1.2  X  10'3 
1  X  1(T3 

1.6 

1.0 
3 

1.6 
1.6 

5  X  1(T' 

2.2  X  10"5 
5.0  X  10"5 

3.7  X  10"7c 
1  X  10~7 

6  X  10"7 
3.7  X  10"7 

1.25 
3.0 


15,800 
7,600 


600-5000 
x  =  1600 
1,580 
1,000 
190-625 
125-625 
150 


3,400 
4,000 

21,600 

25,000 

9,700 

2,300 

980 

370 


2,600 
1,200 


600-5000 

x  =  1600 

1,560 

1,170 

190-625 

125-625 

110 


370 
530 


470 

400 

240 

50 


920 
430 


202 


JON  MALLATT 


TABLE  I — (Continued) 


Animal 


Wet  weight 
(g) 


Flow  rate 
(ml/g/h) 


Flow  rate 

adjusted  to  1.6  g 

wet  body  mass3 


6.   Bryozoan 

Zoobotryon  verticillatum 


5.5  X  10 


5 


6,700 


290 


7.  Ciliates 

a.  Algavores  (large  cells)  e.g., 

Stylonychia  mytilius  5  X  10~8 

b.  Feeders  on  intermediate-sized 

cells  (2-5  jim  diam)  e.g., 

Paramecium  2.5  X  10~8 

c.  Bacterivores  e.g., 

Tetrahymena  pyriformes  1  X  1 0~8 


500,000 

24,000 
5,000 


1,300 

270 
45 


8.  Infaunal  Polychaetes,  and  other 

burrowing  worms 

a.  Sabellidae 

Myxicola  infundibulum 
Schizobranchia  ins  ignis 
Sabella  pavonina 

b.  Serpulidae 

Potamoceras  triqueter 
Hydroides  norvegica 
Spirorbis  borealis 
Salmacina  dysteri 

c.  Chaetopterus  variopedantus 

d.  Urechis  caupo  (Echiuroidea) 

9.  Chordates 

a.  Various  tunicates  (7  genera) 

b.  Branchiostoma  lanceolatum 

c.  Hyla  crucifer  (frog  tadpole) 

d.  Bufo  woodhousei  (frog 


2.7 
1.0 
1.9  X  10- 


io-2 
io-4 


1.9  X 

1.2  X 

2  X 

1  X 

6 

21 


1.6 

1.5  X  IO"2 
0.2 


100 

70 

390 

1,400 
900 
950 

2,090 

50 

900 


95-560,  x  =  225 

200-316 

25-65 


115 

60 

230 

460 

260 

100 

190 

70 

1,230 


95-560,  x  =  225 
70-100 
15-40 


tadpole) 

0.15 

50-140 

30-80 

e.   Larval  Petromyzon  marinus 

1.6 

8-64,  x  =  28 

(in  tube) 

SOURCES:  la.  Paffenhofer  (1976),  Fig.  3;  Ib.  Runge  (1980),  Table  3  (September  value);  2a. 
Mohlenberg  and  Riisgard  (1979);  2b.  Palmer  (1980),  Table  III;  2c.  J0rgensen  (1966);  Fig.  1.40;  2d,e. 
Foster-Smith  (1975),  Figs.  1,2;  2f.  Walz,  (1978),  Fig.  2;  3a.  Crowley  (1973);  3b.  Ryther  (1954),  Figs. 
2,4;  4.  Bogdan  et  al.  (1980),  p.  74-75;  5a,b.  Foster-Smith  (1976),  Table  IV;  6.  Bullivant  (1968);  7. 
Fenchel  (1980a),  p.  18  and  Fig.  4;  8.  Jergensen  (1966),  Table  1.1  and  p.  11;  9a.  Randlov  and  Riisgard 
(1979),  Fig.  4;  9b.  Azariah  (1969);  9c,d.  Scale  and  Beckvar  (1980);  9e.  present  study. 

NOTE:  Essential  data  on  food  type  and  experimental  temperature  are  as  follows:  la.  Various  algae 
at  a  range  of  concentrations,  15°C;  2a.  Various  unicellular  algae,  2  to  10  X  IO4  cells/ml,  10-13°C;  2b. 
Thalassiosira,  Isochrysis  and  Dunaliella,  21  °C;  2c.  "Flagellates  and  diatoms",  22-26°C;  2d,e.  No 
information  given;  2f.  Nitschia  actinastroides,  0-24  mg/1,  15°C;  3a,b.  Rhodotorula  sp.,  20°C;  4a,c,d. 
Chlamydomonas,  20°C;  4b.  Rhodotorula.  20°C;  5a,b.  No  information  given;  6.  Monochrysis,  24°C; 
7.  See  legend  to  Fenchel's  (1980a)  Fig.  4;  8a-c.  Colloidal  graphite  particles,  16-20°C;  8d.  Direct 
measurement,  20°C;  9a.  See  legend  to  Fig.  4  in  Randlov  and  Riisgard  ( 1979);  9b.  "Normal  sea  water", 
29°C;  9c,d.  Anabaena  sphaerica,  0.2-20  mg/1,  21°C;  9e.  Unfed,  or  fed  on  Saccharomyces  cerevisiae, 
12.5°C. 

a  Assumes  F  oc  W075:  See  Materials  and  Methods  section.  Values  also  assume  animals'  dry  weights 
are  20%  of  wet  weight. 

bSee  Winter  (1973,  1978)  and  Foster-Smith  (1976)  for  more  data  on  flow  rates  in  bivalves. 

c  Rotifer  weights  calculated  from  body  lengths  assuming  W  oc  L3  between  genera. 


LARVAL  LAMPREY  FEEDING  203 

be  compared  only  broadly,  as  measurements  reported  in  the  literature  were  obtained 
by  different  methods  and  at  different  temperatures  (but  mostly  between  12  and 
22°C).  An  attempt  was  made  to  correct  for  the  most  important  source  of  variation 
in  published  values  of  flow  per  body  mass  (F/W),  the  effect  of  animal  size.  This 
was  done  through  assuming  FaW075,  i.e.,  F/WaW"025.  Studies  on  a  variety  of 
suspension  feeders  support  this  assumption  (Azariah,  1969;  Paffenhofer,  1971;  J0r- 
gensen,  1975;  M0hlenberg  and  Riisgard,  1979;  Fenchel,  1980a;  Palmer,  1980), 
and  Winter  (1978)  discussed  it  at  length.  In  constructing  the  table,  it  was  also 
necessary  to  assume  dry  weights  of  the  animals  were  20%  of  wet  weight. 

Retention  of  Chlorella  pyrenoidosa  cells  was  measured  for  P.  marinus  am- 
mocoetes.  The  algae  (diameter:  x  =  8.7  ±  2.0  yum  S.D.)  were  grown  in  High  Salt 
Medium  (Sueoka,  1960),  then  washed  via  centrifugation  and  resuspension  in  fil- 
tered tap  water.  Control  experiments  indicated  algal  numbers  did  not  measurably 
increase  during  experimental  periods,  presumedly  because  of  low  light  levels.  Per- 
cent efficiency  of  particle  retention  was  assessed  by  two  techniques.  In  both,  algae 
were  added  to  the  anterior  chamber  of  the  tube  device.  In  the  first  technique,  the 
algal  concentration  was  held  constant  for  an  interval  during  which  a  deflated  bal- 
loon, fitted  around  the  posterior  end  of  the  tube,  collected  the  exhalent  water.  The 
balloon  was  never  allowed  to  fill  to  the  level  where  it  exerted  back  pressure  on  the 
larva.  Samples  were  removed  from  the  balloon  and  the  anterior  compartment, 
diluted  4:1  with  0.1%  NaCl,  and  their  particles  were  counted  six  times  with  a 
Model  FN  Coulter  Counter.  Retention  efficiency  was  calculated  as: 


where  Cb  and  Ca  are  the  numbers  of  algae  counted  per  ml  from  the  balloon  and 
anterior  compartment  samples,  respectively. 

In  the  second  technique,  no  balloon  was  used.  Here,  the  algal  concentration  in 
the  anterior  compartment  decreased  as  the  volume  pumped  from  it  by  the  am- 
mocoete  was  replaced  experimentally  with  clean  water.  Retention  efficiency  was 

calculated  by  the  equation: 

C   V     —  C   V 

r>  T7          ^fp     fp         ^-'ip     tp 

"  ciav,a  -  cfavfa 

^^  id     id         ^^  i  d     id. 

where  Cia  and  Cfa  are  initial  and  final  particle  concentrations  respectively  in  the 
anterior  compartment,  Cip  and  Cfp  are  initial  and  final  particle  concentrations  re- 
spectively in  the  posterior  compartment,  V,a  and  Vfa  are  initial  and  final  volumes 
of  suspension  respectively  in  the  anterior  compartment,  and  Vlp  and  Vfp  are  initial 
and  final  volumes  in  the  posterior  compartment.  Again,  particle  concentrations 
were  measured  with  the  Coulter  Counter. 

Retention  efficiency  was  calculated  a  total  of  22  times,  based  on  1  individuals, 
for  algal  concentrations  ranging  from  1  to  15  mg  (dry)  per  liter. 

RESULTS 

Ammocoetes  in  the  tubes  usually  remained  still,  wiggling  ("crawling",  Rovainen 
and  Schieber,  1975)  being  infrequent.  All  the  individuals  exposed  to  Chlorella 
produced  green  feces  about  six  hours  after  the  presentation  of  food.  Ammocoetes 
dug  from  the  sand  following  exposure  to  yeast  contained  white  cords  within  their 
guts,  visible  through  the  ventral  skin.  These  findings  suggest  the  larvae  fed  normally 
under  experimental  conditions.  Burrows  and  tubes  were  never  lined  by  mucus  (cf. 
Sterba,  1953). 


204 


JON  MALLATT 


100, 


80 


60 


40 


20 


o 
o 


CHLORELLA 
o 

o° 
o   o 


10 


20 


50 


100 


CONC.,  particles  counted /ml, 

FIGURE  3.  Percentages  of  Chlorella  pyrenoidosa  cells  retained  by  larval  P.  marinus  over  a  range 
of  cell  concentrations.  Data  pooled  from  seven  ammocoetes.  A  count  of  2.25  X  105  particles/ml  cor- 
responded to  27  mg  dry  mass  per  liter.  The  open  dots  represent  values  measured  by  collecting  pharyngeal 
efflux  in  a  balloon,  while  the  closed  dots  were  obtained  by  the  dilution  method  described  in  the  text.  The 
least  squares  equation  (linear)  for  all  the  points  is: 

RE  =  86  -  2.94  X  1(T6  C,         r  =  -0.33,         /)>0.10. 


Retention  efficiency  data  are  depicted  in  Figure  3.  For  concentrations  of  Chlo- 
rella between  1  and  75  mg/1,  the  fraction  of  particles  removed  was  high,  averaging 
86  ±  13%  S.D.  and  75  ±  13%  S.D.  respectively,  as  measured  by  the  balloon  and 
dilution  techniques.  These  means  did  not  differ  statistically  from  one  another  at 
the  95%  confidence  level  (/  =  1.96,  P  >  0.05),  so  the  overall  retention  efficiency 
was  calculated  as  82  ±  14%  S.D.  There  was  no  evidence  that  retention  efficiency 
varied  with  algal  concentration  in  the  range  studied. 

Flow  rates  recorded  for  ammocoetes  in  the  tubes  are  shown  in  Figure  4.  One 
hundred  and  one  hourly  recordings,  compiled  from  twelve  individuals,  ranged  from 
8  to  64  ml/g/h.  The  overall  average  hourly  flow  rate  was  28  ml/g/h,  with  a 
standard  deviation  of  13.  Individual  average  flow  rates  during  the  monitoring  pe- 
riods ranged  from  10  to  52  ml/g/h  in  the  twelve  animals.  Did  flow  rate  tend  to 
change  with  the  amount  of  time  spent  in  the  tube?  When  both  increases  and 
decreases  are  considered,  the  mean  hourly  change  did  not  differ  significantly  from 
zero  (+3.2%  ±  28%  S.D.,  P  >  0.3).  Absolute  hourly  changes  averaged  22%.  Thus, 
although  flow  rates  varied  considerably  over  time,  no  consistent  pattern  of  variation 
was  evident. 

Clearance  rates  recorded  for  burrowed  ammocoetes  are  depicted  in  Figure  5. 
The  quite  similar  results  from  the  two  species  were  combined.  Mean  clearance  rates 
ranged  from  3  to  13  ml/g/h,  depending  on  the  concentration  of  yeast  in  the  over- 
lying water,  with  an  overall  average  of  about  7  ml/g/h. 

The  placement  of  a  4-cm  sand  plug  in  the  path  of  pharyngeal  efflux  of  three 
ammocoetes  in  tubes  led  to  decreases  in  flow  over  previous  rates.  Declines  averaged 
about  50%  (30  to  11,  23  to  15,  and  19  to  11  ml/g/h). 

DISCUSSION 

It  could  be  suggested  that  confining  ammocoetes  within  tubes  affected  flow  rate, 
either  physically — the  glass  walls  interfering  with  movement  of  water  out  of  the 


LARVAL  LAMPREY  FEEDING 


205 


O) 
\ 

•^•B 

E 


O 


90 
80 
70 
60 
50 
40 
30 
20 
10 


90 
80 
70 
60 
50 
40 
30 
20 
10 


10         12 


14 


1O          12 


14 


TIME,  hours 

FIGURE  4.  Variation  in  flow  rate  over  time  in  twelve  nonfeeding  P.  marinus  larvae  (x  =  1.6  g), 
each  within  the  device  of  Figure  2,  at  12°C.  Two  graphs  are  used  to  avoid  crowding.  No  consistent 
pattern  of  change  is  evident. 


u 

z 


LU 

u 


50 

30 
20 

10- 

5 

3 
2 


8 


13 


235   10  20  30  50   100  200    500  1000 


5000 


CONCENTRATION,  mg/l 


FIGURE  5.  Response  to  yeast  cells  (Saccharomyces  cerevisiase)  of  ammocoetes  (x  =  2.0g)  bur- 
rowed in  sand,  particle  clearance  rate  vs.  food  concentration  in  the  overlying  water.  Data  from  two 
species  P.  marinus  and  L.  tridentatus  were  very  similar  (analysis  of  covariance,  P  >  0.25  for  both  slopes 
and  intercepts  of  the  log-transformed  lines),  and  are  combined.  The  least  squares  equations,  calculated 
from  the  log-transformed  data,  are:  for  Petromyzon,  F  =  2.9 -C017  (46  points,  r  =  0.29,  P  =  0.05);  for 
Lampetra,  F  =  2.3-C025  (43  points,  r  =  0.37,  P  <  0.05);  and  for  the  combined  data,  as  graphed,  F 
=  2.3-C021  (r  =  0.33,  P  <  0.01).  The  dots  represent  mean  values  for  points  in  the  concentration  ranges 
indicated  by  the  horizontal  bars.  Vertical  bars  delineate  95%  confidence  intervals  for  clearance  rates, 
and  the  numbers  of  points  used  in  calculating  these  intervals  are  indicated  below  these  bars.  At  12°C. 


206  JON  MALLATT 

gill  pores — or  behaviorally,  through  stressing  the  animals.  Speaking  against  phys- 
ical interference,  it  is  noted  that  flow  from  the  gill  pores  is  normally  posterior,  not 
lateral  (unpublished  observations,  on  free  animals  presented  concentrated  carmine 
particle  suspensions.).  Speaking  against  stress,  it  is  noted  that  larvae  in  tubes  ef- 
ficiently ingested  food  (Fig.  3),  and  seldom  exhibited  crawling  behavior.  The  lack 
of  any  direction  of  variation  in  flow  rate  with  time  (Fig.  4)  is  also  consistent  with 
the  view  that  the  ammocoetes  were  not  stressed.  Indeed,  placement  in  a  tube  seems 
to  calm  this  burrowing  organism  (thigmokinesis:  Hardisty,  1979,  p.  56;  the  calming 
effect  was  also  noted  by  Rovainen  and  Schieber,  1975). 

Rovainen  and  Schieber  (1975)  validly  point  out  that  such  tubes  may  interfere 
with  cutaneous  respiration,  leading  to  a  compensatory  elevation  of  flow  through 
the  pharynx.  The  degree  to  which  ammocoetes  rely  on  cutaneous  respiration  is 
unknown  (Lewis,  1980),  although  the  thickness  of  the  dermis  suggests  that  the  gills 
are  much  more  important  respiratory  structures  than  the  skin  (Czopek  and  Sawa, 
1971 ).  Furthermore,  opportunity  for  cutaneous  respiration  should  be  curtailed  when 
ammocoetes  occupy  poorly  permeable  substrate.  An  overestimate  of  flow  rates 
would  not  affect  the  conclusions  of  this  paper. 

The  test  of  retention  efficiency  employed  here  measured  the  fraction  of  particles 
removed  from  suspension,  not  the  fraction  that  actually  entered  the  gut.  It  is  con- 
ceivable that  some  error  was  introduced  through  the  ammocoetes  rejecting  particles 
after  filtration,  or  by  some  cells  settling  within  the  tubes  and  never  reaching  the 
balloon  (although  nothing  was  seen  that  indicated  these  things  occurred).  In  future 
studies,  the  methodology  will  be  expanded  to  include  a  quantification  of  gut  con- 
tents. 

Malmqvist  and  Bronmark  (1982)  determined  clearance  rates  of  Lampetra  pla- 
neri  ammocoetes  in  sand.  Using  the  same  technique,  I  obtained  average  clearance 
rates  (7  ml/g/h)  that  are  comparable  to  theirs  (11  ml/g/h),  considering  that 
their  animals  were  smaller  (0.6,  cf.  1.6g)  and  the  temperature,  higher  (15°C). 
However,  those  authors  apparently  considered  their  clearance  rates  to  reflect  flow 
rates,  which  may  not  be  correct.  In  the  technique  employed,  a  mass  of  clean  water, 
within  the  pore  space  of  the  sediment,  is  interposed  between  the  ammocoetes  and 
the  overlying  suspension.  To  the  extent  that  the  ammocoetes  use  this  clean  water, 
particles  will  not  be  cleared,  and  clearance  rates  will  underestimate  flow  rates.  That 
the  discrepancy  is  significant  is  indicated  by  the  observation  that  most  ammocoetes 
in  the  test  aquaria  had  closed  burrows,  cut  off  from  the  suspension  overhead.  As 
the  flow  rates  of  the  burrowed  ammocoetes  in  this  study  cannot  be  measured  by 
clearance  rates,  they  must  be  estimated  by  simulation.  When  pumping  against  a 
sand  plug,  whose  length  approximated  the  depth  at  which  burrowed  ammocoetes 
reside,  larvae  in  tubes  moved  water  at  about  half  their  unimpeded  rate;  thus,  flow 
rates  for  the  burrowed  animals  in  this  study  are  estimated  as  half  those  of  unbur- 
rowed  individuals,  or  about  15  ml/g/h. 

In  this  study,  ammocoetes  filtered  most  (x  =  82%)  Chlorella  particles  from 
suspension  over  a  range  of  concentrations,  1-75  mg/1,  that  should  include  those 
they  experience  in  nature.  Many  suspension  feeders  begin  to  perform  inefficiently 
when  concentrations  exceed  1-10  mg/1,  rejecting  particles  (J0rgensen,  1975,  pp. 
64-65;  Epifanio  and  Ewart,  1977).  The  evidence  for  efficient  retention  by  am- 
mocoetes at  concentrations  as  high  as  75  mg/1  supports  the  hypothesis,  proposed 
in  the  Introduction,  that  lamprey  larvae  are  adapted  to  filter  concentrated  suspen- 
sions. The  extensive  system  of  feeding  mucus  may  allow  this. 

Average  flow  rates,  as  measured  for  the  animals  in  tubes,  varied  among  indi- 
viduals by  a  factor  of  five  (10-52  ml/g/h).  This  large  variation  is  noteworthy, 


LARVAL  LAMPREY  FEEDING 


207 


^ 
7         8         9        10        »        \Z        13       \«       \S       \ft 


FIGURE  6.  Photographs  demonstrating  that  ammocoetes  propel  exhalent  ventilatory  water  into  the 
surrounding  sediment.  Above  is  shown  an  empty  burrow  (e)  against  the  wall  of  an  aquarium;  below  are 
two  burrows  containing  ammocoetes  (a).  Most  of  the  sand  is  dark,  containing  reduced  organic  matter. 
Above,  a  thin  rim  of  light,  oxidized  sand  outlines  the  empty  burrow.  This  demonstrates  the  low  per- 
meability of  the  sand,  aerated  water  having  only  diffused  a  few  mm  into  it.  Below,  both  ammocoetes 
(a)  are  surrounded  by  light  halos,  1-2  cm  thick,  produced  by  their  pharyngeal  efflux.  Such  halos  also 
surrounded  ammocoetes  buried  in  a  very  fine  grained  diatomaceous  earth,  although  the  light  zones  were 
thinner  there.  Oxidized  zones  also  have  been  noted  around  infaunal  deposit  feeders  (Aller,  1978, 
Fig.  1). 


considering  animal  weights,  temperature,  and  treatments  were  all  standardized. 
Rovainen  and  Schieber  ( 1 975,  their  Table  1 )  also  recorded  large  individual  variation 
in  flow  rates;  these  ranged  from  20  to  60  ml/g/h  for  undisturbed  ammocoetes  at 
20°  weighing  0.85-2.7  g. 

In  the  present  study,  flow  rates  were  calculated  to  average  28  and  about  15 
ml/g/h  for  unburrowed  and  burrowed  ammocoetes  (x  =  1.6  g,  12°).  Preliminary 
results  from  similar  animals  in  tubes  indicate  that  the  presence  of  food  (yeast)  can 
increase  flow  rate  by  up  to  50%,  to  about  50  ml/g/h  (Mallatt,  1980).  Even  so, 


208  JON  MALLATT 

ammocoete  flow  rates  are  probably  the  lowest  ever  recorded  for  a  suspension  feeding 
animal,  even  when  adjusted  to  compensate  for  the  ammocoete's  comparatively  large 
size  (Table  I,  fourth  column).  The  low  flow  rates  of  ammocoetes  are  more  like 
those  produced  by  animals  that  do  not  depend  on  suspension  feeding,  such  as 
macrophagous  fish  (Randall,  1970),  infaunal  deposit  feeders  (Echinocardium  and 
Malacoceras:  Foster-Smith,  1978),  and  some  facultative  suspension  feeders  (Ar- 
enicola  and  Bithynia:  J0rgensen,  1966). 

Several  factors  could  be  responsible  for  the  low  rate  at  which  ammocoetes  pump 
water.  Most  obviously,  this  should  relate  to  the  resistance  of  the  substrate  inhabited, 
which  would  preclude  the  evolution  of  a  rapid  flow  rate.  The  quantity  of  intra- 
pharyngeal  mucus  might  also  limit  ammocoete  flow  rate,  as  do  the  fine  mesh  filters 
of  some  holotrich  ciliates  (Fenchel,  1980a,b). 

This  analysis  reveals  two  peculiarities  of  ammocoete  feeding.  Compared  to  other 
suspension  feeders,  ammocoetes  1)  pump  water  extremely  slowly,  and  2)  are  able 
to  filter  very  concentrated  suspensions.  These  are  interrelated.  Slow  flow  allows 
concentrated  suspensions  to  be  utilized  in  that,  by  presenting  little  food-carrying 
water  to  the  filtering  surfaces  per  unit  time,  it  diminishes  the  tendency  of  these  to 
saturate.  Low  flow  rate  also  demands  high  food  concentration,  for  only  concentrated 
suspensions  could  be  expected  to  fill  nutrient  needs  when  little  food-carrying  water 
is  available  per  unit  time. 

An  hypothesis  of  the  ammocoete  feeding  strategy  emerges  from  this  analysis. 
Whereas  most  suspension  feeders  meet  their  food  requirement  by  moving  dilute 
suspensions  rapidly  across  their  feeding  structures,  ammocoetes  cannot  grow  on 
dilute  suspensions.  A  slow  rate  of  water  flow  through  the  pharynx,  necessitated  by 
the  high  resistance  of  the  substrate  inhabited  and  the  design  of  the  pharyngeal 
pump,  confines  ammocoetes  to  environments  where  food  suspensions  are  concen- 
trated. Since  the  burrowing  habit  that  limits  flow  rate  is  necessary  for  protecting 
lampreys  from  predation  during  the  larval  stage  (Morman  et  al.,  1980),  the  re- 
quirement for  concentrated  suspensions  seems  basic  to  the  animal's  biology. 

The  peculiarities  of  ammocoete  feeding  could  be  of  general  ecological  interest. 
The  ability  of  infaunal  animals  to  modify  the  chemistry  of  the  substrate  they  inhabit 
has  recently  received  much  attention  (Aller,  1980;  Gust  and  Harrison,  1981;  Law- 
rence et  al.,  1982).  For  ammocoetes,  which  drive  the  overlying  water  directly  into 
the  sediment  (Fig.  6),  habitat  modification  could  be  considerable. 

ACKNOWLEDGMENTS 

This  work  was  partly  supported  by  the  Basic  Medical  Sciences  Program  ( WAMI) 
at  Washington  State  University;  part  was  performed  at  the  University  of  Chicago, 
Dept.  of  Anatomy,  under  the  support  of  the  University's  Hinds  and  Nierman  Funds. 
I  would  like  to  thank  Kwen-Scheng  Chiang,  J.  J.  Gilpin,  James  Huber,  Timothy 
Holtsford,  Aniko  Juhasz,  Natasha  Matkin,  David  Swinton,  Richard  Wallace,  and 
Richard  Wassersug  for  help  with  various  aspects  of  this  study.  Martin  Feder,  Bruce 
Frost,  Karin  Hoff,  G.  R.  Harbison,  Mike  LaBarbera,  J.  T.  Lehman,  Richard  Parker, 
Dianne  Scale,  and  Richard  Wassersug  critically  read  various  versions  of  the  manu- 
script. 

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Reference:  Biol.  Bull.  163:  211-224.  (August,  1982) 


POST-LARVAL  GROWTH  OF  DISSODACTYLUS  PRIMITIVES 

BOUVIER,  1917  (BRACHYURA:  PINNOTHERIDAE)  UNDER 

LABORATORY  CONDITIONS 

GERHARD  POHLE  AND  MALCOLM  TELFORD 
Department  of  Zoology,  University  of  Toronto,  Ontario,  Canada  M5S  1A1 

ABSTRACT 

Dissodactylus  primitivus  is  a  small  pinnotherid  crab  parasitic  on  spatangoid 
urchins.  Post-larval  growth  has  been  observed  in  the  laboratory  in  the  absence  of 
hosts.  Individual  animals  were  grown  over  the  whole  size  range  of  the  species  for 
a  period  of  691  days  after  hatching.  During  growth  of  D.  primitivus  males  and 
females,  there  was  no  significant  change  in  carapace  width  with  length.  Relative 
to  carapace  width,  male  abdominal  width  increased  isometrically.  Growth  of  the 
female  abdomen  was  allometric  and  could  not  be  explained  by  a  simple  relationship. 
Two  phases  leading  to  sexual  maturity  were  recognized:  one  of  low  positive  al- 
lometry,  the  other  of  strong  allometric  growth.  During  the  latter  phase  pubertal 
molts  occurred. 

Growth  over  time  showed  decreasing  increments  and  an  increase  in  intermolt 
periods,  with  slight  differences  between  sexes.  The  resulting  growth  rates  closely 
fitted  power  curves.  Compared  to  females,  growth  of  males  decreased  after  the  first 
year.  This  could  explain  the  presence  of  larger  females  in  natural  populations. 

Several  growth  relationships  analogous  to  weight  were  demonstrated  and  the 
results  discussed  in  relation  to  other  Crustacea. 

INTRODUCTION 

Studies  of  age  and  growth  contribute  to  an  understanding  of  population  dy- 
namics and  to  the  elucidation  of  developmental  processes.  Among  Crustacea,  such 
studies  have  most  often  centered  on  species  of  commercial  interest  (Maucheline, 
1977),  including  prawn  (Forster,  1970;  Wickins,  1976),  lobster  (Thomas,  1965; 
Ennis,  1972)  and  crabs  (Weber,  1967).  Little  comparable  information  is  available 
on  crustacean  species,  especially  crabs,  in  the  low  size  range.  Dissodactylus  prim- 
itivus is  a  small  pinnotherid  crab  with  a  maximum  carapace  width  of  less  than  1 
cm,  living  as  a  parasite  on  the  spatangoid  urchins  Meoma  ventricosa  and  Pla- 
giobrissus  grandis  (Telford,  1978b,  1982). 

Despite  many  attempts  since  the  landmark  paper  by  Kurata  (1962)  to  com- 
prehensively describe  age  and  growth  in  crustaceans,  one  major  problem  has 
persisted.  It  is  the  difficulty  of  obtaining  morphometric  data  for  known-age  indi- 
viduals. Several  studies  of  allometry  have  relied  entirely  on  collections  of  wild 
specimens  of  unknown  age.  For  example,  Finney  and  Abele  (1981)  have  analyzed 
changes  of  shape  with  size  in  a  xanthid  crab,  Trapezia  ferruginea  and  Williams 
et  al.  (1980)  have  compared  size  and  shape  relationships  in  three  species  of  Uca. 

Received  5  March  1982;  accepted  24  May  1982. 

Abbreviations:  ABDW,  abdominal  width;  AGE,  days  after  hatch;  CL,  carapace  length;  CW,  car- 
apace width;  INCR,  increment;  INSTAR,  instar  number;  INTMT,  intermolt  period;  P-INCR,  percent 
increment. 

211 


212  G.  POHLE  AND  M.  TELFORD 

Similarly,  Haley  (1969)  was  able  to  correlate  sexual  maturity  with  external  mor- 
phology in  the  ghost  crab,  Ocypode  quadrata,  from  specimens  of  unknown  age. 
Growth  increments  at  successive  molts  have  also  been  estimated  from  individuals 
of  unknown  age.  Thus  Sheader  (1981)  described  increase  in  size  in  an  amphipod, 
Parathemisto  gaudichaudi,  from  wild  specimens  maintained  in  the  laboratory.  Data 
from  tagged  animals  released  and  recaptured  (e.g.  Bennet,  1974)  suffer  from  the 
same  inherent  uncertainty.  Theoretically,  the  solution  to  the  problem  should  be 
simple:  culture  the  organisms  from  egg  or  known  larval  instars.  With  known-age 
individuals  it  is  possible  to  analyze  the  two  principal  components  of  growth  (Need- 
ham,  1964),  namely,  rate  and  change  of  form  (differential  growth).  Although  con- 
siderable success  has  been  achieved  in  laboratory  culture  of  shrimps  and  lobsters 
(Bardach  et  al,  1972),  few,  if  any,  crabs  have  been  successfully  reared  in  continuous 
culture. 

We  have  raised  Dissodactylus  primitivus  from  egg  to  adult  size,  providing  a 
series  of  measurements  for  each  of  the  thirty  or  more  instars  for  individuals  of 
precisely  known  age.  Post-larval  growth  is  examined  here  with  two  major  objectives: 
the  first  to  observe  changes  in  form  with  reference  to  size,  the  second  to  investigate 
growth  as  it  occurs  over  time.  Larval  development  is  described  elsewhere  (Pohle 
and  Telford,  1983).  This  paper  on  post-larval  growth  thus  completes  a  develop- 
mental study  of  D.  primitivus. 

MATERIALS  AND  METHODS 

Collection  of  parental  stock.  Ovigerous  female  crabs  were  collected  with  host 
spatangoid  urchins,  Meoma  ventricosa,  from  sandy  bottoms  in  5-18  m  of  water, 
off  the  western  coast  of  Barbados.  Specimens  were  kept  in  50-liter  tanks  until 
hatching  occurred.  Egg  masses  were  periodically  examined  for  maturity  and  pos- 
sible protozoan  or  fungal  infection.  Newly  produced  egg  masses  are  bright  orange 
in  color,  becoming  pale  yellow  towards  the  end  of  maturation.  Hatching  of  zoeae 
for  this  study  occurred  between  9  and  11  p.m.  on  June  12,  1978. 

Rearing  procedure.  At  time  of  hatching  a  numbered  series  of  100  of  the  most 
vigorously  swimming,  positively  phototactic  larvae  were  pipetted  into  individual 
120-ml  glass  jars  2/3  filled  with  sea  water.  Maintenance  of  larvae  followed  procedures 
outlined  by  Pohle  and  Telford  (1983).  Post-larvae  were  inspected  daily,  given  live 
food,  and  transfered  to  new  containers  with  about  90  to  120  ml  fresh  sea  water 
(depending  on  size).  Measurements  were  taken  following  every  molt.  Animals  and 
containers  were  kept  in  a  water  table  of  running  sea  water  to  approximate  tem- 
peratures to  natural  conditions. 

Culture  medium  and  conditions.  Sea  water  fed  into  the  laboratory  was  found 
to  be  inadequate  even  when  filtered.  Instead,  fresh  sea  water  was  collected  daily 
about  500  m  offshore.  In  this  way  filtering  or  addition  of  antibiotics  were  found 
to  be  unnecessary. 

For  the  entire  rearing  salinity  ranged  from  31.5  to  34%o  (mean  33%o).  Tem- 
perature varied  between  26.5  and  29.5°C  (mean  28°C),  about  0.5  to  1.0°C  above 
the  natural  environment.  A  14-h  photoperiod  was  maintained. 

Food.  Larvae  and  early  post-larvae  were  fed  with  newly  hatched  Anemia  nauplii 
ad  libitum.  In  addition,  selected  plankton  of  appropriate  size,  collected  in  daily 
trawls,  was  given  as  a  food  supplement.  For  later  stages  increasingly  larger  Anemia 
nauplii  and  planktonic  organisms  on  which  crabs  readily  fed  were  used. 

Measurements.  At  each  instar,  specimens  were  measured  live,  twice,  and  the 
results  averaged.  After  molting  the  exuvium  was  also  measured.  No  significant 
difference  was  found  between  exuvial  and  live  measurements. 


D.  PRIMITIVUS  POST-LARVAL  GROWTH  213 

Imminence  of  molting  was  apparent  by  a  change  in  carapace  opacity  1-2  days 
before  exuviation.  Exuviae  were  never  eaten  by  any  of  the  specimens. 

The  following  measurements  were  made  under  the  light  microscope  by  cali- 
brated ocular  micrometer:  maximum  carapace  width,  dorsally  and  anteriorly;  car- 
apace length  from  vestigial  rostrum  to  posterior  margin,  ignoring  curvature;  and 
width  of  abdomen  at  its  widest  point. 

Statistical  analysis.  All  regression  lines  were  fitted  by  least  squares  analysis. 
Its  major  disadvantage  is  that  error  is  assumed  to  occur  in  only  one  of  the  two 
variates  and  may  result  in  a  low  estimate  of  slope  (Gould,  1975).  There  are  several 
other  methods  (Sokal  and  Rohlf,  1969)  which  consider  error  in  both  variates,  but 
least  squares  was  used  here  following  arguments  given  by  Brown  and  Davies  (1972), 
Gould  (1966),  and  Finney  and  Abele  (1981):  (1)  it  is  easier  to  interpret  and  allows 
the  use  of  standard  tests  of  significance;  (2)  since  most  correlation  coefficients  (r) 
in  this  study  are  above  0.90,  results  with  other  methods  should  not  be  substantially 
different;  and  finally,  (3)  a  comparison  of  methods  by  Brown  and  Davies  (1972), 
using  Doryline  ants,  has  shown  differences  in  results  to  be  very  small.  This  was  at 
least  partly  attributed  to  the  particular  discontinuous  growth  pattern  of  the  ar- 
thropod exoskeleton,  where  size  differences  between  instars  are  much  greater  than 
for  individuals  of  a  given  instar.  The  same  argument  applies  here. 

Zar  (1968)  raised  objections  to  the  widely  accepted  use  of  log  transformations 
of  power  functions,  suggesting  instead  the  use  of  the  curvilinear  non-transformed 
model.  This  problem  has  not  yet  been  satisfactorily  resolved  (Finney  and  Abele, 
1981 ),  but  the  conventional  linear  transformation  used  here  has  been  recommended 
(Sacher,  1970). 

In  order  to  study  changes  in  growth,  a  reference  dimension  which  itself 
shows  little  or  no  change  in  growth  rate  is  selected  (Brown  and  Davies,  1972).  In 
brachyuran  crustaceans  this  is  usually  either  carapace  length  (e.g.  Finney  and 
Abele,  1981)  or  carapace  width  (e.g.  Barnes,  1968).  A  regression  of  these  two 
parameters  is  often  isometric  (Warner,  1977),  i.e.  without  significant  change  in 
ratios  during  growth.  Width  was  chosen  here  because  carapace  length  in  D.  prim- 
itivus  is  a  less  reliable  measurement  due  to  curvature  along  the  longitudinal  axis. 

The  power  function  y  —  axb  is  known  to  biologists  as  the  equation  of  simple 
allometry.  It  has  found  wide  application  in  the  analysis  of  growth  (Gould,  1966). 
The  theoretical  basis  claimed  for  this  function  by  Teissier  (1960),  however,  has 
not  been  universally  accepted  (Kidwell  and  Williams,  1956),  and  consequently  it 
should  not  be  considered  a  fundamental  law  of  growth  (Pasternack  and  Gianutsos, 
1969).  In  this  study  linear  (y  =  bx  +  a),  semi-log  (log  y  =  bx  +  a),  and  power 
functions  (as  log-log,  log  y  =  b-log  x  +  log  a)  were  applied  to  all  data  and  that 
model  which  combined  the  simplest  explanation  of  the  data  with  the  best  possible 
fit  was  chosen. 

For  determination  of  allometric  status,  regressions  were  tested  against  either 
an  isometric  intercept  standard  of  0  for  linear  regression,  or  an  isometric  slope 
standard  of  1  for  power  functions  with  a  Students  Mest  (Sokal  and  Rohlf,  1969). 
Analysis  of  co-variance  (F-test)  was  employed  to  compare  slopes.  Regression  lines, 
statistics,  and  bivariate  scattergrams  were  obtained  by  computer  from  programs 
in  the  Statistical  Analysis  System  (SAS)  package. 

RESULTS 
Survival  and  mortality 

In  the  laboratory  mortality  was  highest  during  the  relatively  short  larval  life, 
especially  the  megalopa,  only  44  reaching  the  first  crab  instar  (Fig.  1 ).  The  number 


214 


G.  POHLE  AND  M.  TELFORD 


DC 

o 


100 


80 


OC      60 

D 

CO 


o 

QC 


40 


20 


-Z3 

I-M 


100 


200 


300 


400 


500 


600 


TIME    (days) 


FIGURE  1.  Survival  of  Dissodactylus  primitivus  in  the  laboratory.  Points  represent  mean  values 
for  successive  instars.  Symbols:  A,  zoeal  stages  (Zl  to  Z3);  •,  megalopa  (M);  •,  post-larval  instars  (Cl 
to  C28). 

of  survivors  then  gradually  decreased,  to  21  by  instar  16  (mean  day  169),  with  no 
further  deaths  occurring  for  about  another  '/2  year  (instar  23,  mean  day  335). 
Subsequently,  numbers  of  survivors  steadily  decreased  (a  constant  average  mor- 
tality rate  represented  by  points  falling  in  a  straight  line)  to  12  by  post-larval  instar 
28,  almost  600  days  after  hatching.  On  day  691  the  rearing  experiment  was  dis- 
continued with  4  survivors  remaining,  2  females  (instars  28,  32)  and  2  males  (instars 
28,  29). 

Carapace  width  and  length 

The  relationship  of  mean  carapace  widths  (CW)  and  lengths  (CL)  for  post- 
larval  instars  1-28  and  of  individual  values  for  two  specimens  which  reached  instars 
30  and  32  (Fig.  2)  was  given  by  the  equation: 

CL  =  0.828  •  CW  +  0.036         (r2  =  0.994) 

This  calculated  line,  for  the  two  sexes  together,  was  based  on  550  pairs  of  mea- 
surements. For  separate  sexes  the  intercepts  were  not  significantly  different  from 
0  (/-statistic  =  1.02,  P  =  0.31  for  males;  /  =  1.81,  P  =  0.07  for  females).  Thus 
the  relationship  was  regarded  as  isometric. 

The  difference  of  slope  between  sexes  was  not  significant  (F  =  0.35,  P  =  0.55). 
Male  crabs  (max  CW  =  8.1  mm),  however,  never  reached  sizes  of  the  largest 
females  (max  CW  =  9.5  mm).  These  large,  laboratory-reared  crabs  are  of  equal 
or  greater  size  than  those  found  in  the  wild  (CW  <  10  mm). 


Carapace  width  and  abdominal  width 

Based  on  239  paired  measurements  of  crabs  from  post-larval  instars  5  to  28, 
the  relationship  of  abdominal  width  (ABDW)  and  carapace  width  (CW)  for  male 
crabs  was: 

ABDW-0.389-CW-0.il?         (r2  =  0.997) 


D.  PRIMITIVUS  POST-LARVAL  GROWTH 


215 


Fitting  the  data  to  a  power  function  resulted  in  the  equation: 

ABDW  =  0.309  •  CW1  °'°         (r2  =  0.998) 
Figure  3  shows  a  log-log  transformation,  where: 

log  ABDW  =  1.010-logCW  --  0.510 

The  slope  of  1.01  indicated  isometric  growth  (/  =  1.30,  P  =  0.21). 

During  growth,  abdominal  width  in  females  increased  more  in  higher  instars, 
fitting  a  power  curve  which,  expressed  as  a  log-log  function,  was  given  by  the 
equation: 

log  ABDW  =  1.481  -log  CW  --  0.616         (r2  ==  0.984,  N  =  226) 

Regression  slopes  were  significantly  different  for  the  sexes  (F  =  1146. 10,  P 
=  0.0001). 

Analysis  of  growth  of  individuals  and  means  for  instars  (Fig.  3)  showed  that 
the  relationship  was  not  of  simple  allometry.  There  was  a  change  of  slope  approx- 
imately between  instars  19  and  20  (see  arrow  Fig.  3).  The  data  were  better  rep- 
resented by  two  separate  regressions,  for  early  instars: 

log  ABDW  =  1 .295  •  log  CW  --  0.548         (r2  =  0.995,  N  =  157) 
and  for  later  instars: 

log  ABDW  ----  2.025- log  CW  --  1.048         (r2  =  0.931,  N  =  69) 

Both  slopes  were  significantly  greater  than  1  (/ =  55.14,  P  =  0.0001  and  / 
=  35.42,  P  =  0.0001 ,  respectively)  and  hence  growth  was  not  isometric  but  positively 
allometric.  The  two  slopes  were  significantly  different  (F  =  541.33,  P  =  0.0001), 
indicating  markedly  different  growth  in  the  two  size  groups. 


E 
E 


O 


LLJ 


QC 


O 


80 


7.0 


6.0 


5.0 


4.0 


O 

a      3.0 


2° 


1.0 


1.0         2.0         3.0         4.0         50         6.0         7.0         8.0 
CARAPACE     WIDTH     (mm) 


9.0       100 


FIGURE  2.  Relative  growth  of  the  carapace  of  Dissodactylus  primitivus.  Each  circle  represents 
a  mean  measurement  for  one  instar  up  to  instar  28.  Number  of  observations  per  instar  is  shown;  if  not 
given,  number  equals  that  of  adjacent  instar.  Vertical  and  horizontal  lines  are  standard  deviations. 
Additional  points  are  given  for  the  lone  male  (A)  and  the  lone  female  (D)  which  surpassed  instar  28. 


216 


G.  POHLE  AND  M.  TELFORD 


CO 


8.0 
6.0 

4.0 


I 

9     2.0 


1.0 
0.8 

0.6 

0.4 
0.3 


+ 


1.0 


2.0  4.0  6.0 

CARAPACE    WIDTH    (mm) 


8.0    10.0 


FIGURE  3.  Relative  growth  of  the  abdomen  of  Dissodactylus  primitivus.  Open  symbols  represent 
males,  closed  symbols  females.  Each  circular  symbol  is  a  mean  measurement  for  one  instar,  with  the 
number  of  observations  given.  Vertical  and  horizontal  lines  are  standard  deviations.  Four  additional 
square  symbols  represent  instars  29  to  32  of  one  female  individual  in  which  molt  of  instar  28  to  29 
represents  a  pubertal  molt.  Arrow  between  instars  19  and  20  indicates  point  of  transition  from  early  to 
late  prepubertal  growth. 


Intermolt  period 

Intermolt  period  (INTMT)  and  carapace  width  (CW)  best  fit  a  semi-log  rela- 
tionship (Fig.  4),  as  follows: 

Males:  log  INTMT  =  0. 1 27  •  CW  +  0.722         (r2  =  0.875,  N  =  260) 
Females:  log  INTMT  ••*  0.110-CW  +  0.767         (r2  =  0.835,  N  =  250) 

Slopes  of  these  regressions  were  significantly  different  (F  =  15.66,  P  =  0.0001). 

A  similar  relationship  was  found  between  intermolt  period  and  successive  post- 
larval  instar  numbers: 

Males:  log  INTMT  =  0.038  •  INSTAR  +  0.671         (r2  =  0.899,  N  =  293) 
Females:  log  INTMT  =  0.034-  INSTAR  +  0.703         (r2  =  0.859,  N  -  279) 


D.  PRIMITIWS  POST-LARVAL  GROWTH 


217 


Slope  differences  between  sexes  were  significant  (F  =  10.03,  P  =  0.0002).  Although 
these  appear  negligible,  intermolt  periods  become  significantly  different  at  higher 
instars.  For  example,  at  instar  7  calculated  intermolt  periods  were  8.6  days  for 
males,  8.7  days  for  females  and  at  instar  27  corresponding  periods  were  49.8  and 
41.8  days.  Regression  equations  for  intermolt  period  and  carapace  width  (above) 
yielded  similar  differences. 

Growth  increments 

Significant  linear  correlation  was  obtained  between  percent  growth  increments 
and  carapace  width  or  instar  number  using  log-linear  and  linear-linear  regressions, 
as  obtained  for  other  Crustacea  by  Maucheline  (1977).  Non-transformed  regres- 
sions were  chosen  here,  however,  because  of  the  significantly  better  fit  for  both 
sexes.  Plotting  percent  increment  (P-INCR)  against  carapace  width  (CW)  (see 
Fig.  5)  resulted  in  the  equations: 


Males:  P-INCR  =  -1.464-CW  +  14.894 
Females:  P-INCR  =  -1.194-CW  +  14.328 


(r2  =  0.709,  N  =  256) 
(r2  =  0.734,  N  =  248) 


An  F  value  of  13.16  indicated  a  difference  of  slope  between  sexes  (P  =  0.0001). 
Semi-log  regression  of  the  same  data  gave  r2  values  of  0.605  and  0.698,  respectively. 
A  similar  relationship  was  obtained  between  percent  increment  and  successive  instar 


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FIGURE  4.  Intermolt  periods  and  carapace  widths  of  Dissodactylus  primitivus.  Symbols  as  in 
Figure  3.  Vertical  bars  represent  standard  deviations.  Additional  intermolt  periods  are  given  for  one 
male  (A,  instars  29-30)  and  one  female  (•,  instars  29-32). 


218 


G.  POHLE  AND  M.  TELFORD 


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FIGURE  5.    Carapace  growth  of  Dissodactylus  primitivus  males  and  females  as  a  percent  increment 
based  on  premolt  width.  Symbols  as  in  Figure  3. 


numbers: 

Males:  P-INCR  =    -0.449- INSTAR  +  15.758         (r  =  0.707,  N  ==  256) 
Females:  P-INCR  =    -0.382- INSTAR  +  15.214         (r  =  0.729,  N  ==  248) 
Semi-log  regression  resulted  in  lower  r2  values  (0.556  and  0.664,  respectively). 

Growth  rate 

The  relationship  between  carapace  width  (CW)  and  age  (days  after  hatch)  is 
shown  in  Figure  6.  For  both  sexes  the  data  best  fit  a  power  function. 

Males:  log  CW  ==  0.739  •  log  AGE  -  1.053         (r2  =  0.970,  N  ==  268) 
and 

log  AGE  ==  1.311- log  CW  +  1.445 

Females:  log  CW  ==  0.791  -log  AGE  -    1.172         (r  =  0.952,  N  =  260) 
and 

log  AGE  ==  1 .203  •  log  CW  +  1 .5 1 2 

Slopes  of  male  and  female  regressions  were  significantly  different  (F  =  14.46, 
P  =  0.0002).  This  seems  to  be  primarily  explained  by  the  decreased  carapace 
growth  of  older  males.  This  is  in  agreement  with  observations  that  D.  primitivus 
adult  males  are  smaller  than  females  in  the  wild.  Growth  in  carapace  width  of 
individual  females  which  passed  through  pubertal  molts  showed  no  significant  de- 
parture from  the  curve  (square  symbols,  Fig.  6).  Thus,  in  contrast  to  abdominal 
width,  carapace  width  and  intermolt  period  does  not  seem  to  change  abruptly  at 
the  onset  of  sexual  maturity. 


D.  PRIMITIWS  POST-LARVAL  GROWTH 


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FIGURE  6.  Growth  rate  of  Dissodactylus  primitivus  males  and  females.  Open  and  closed  circles 
represent  mean  measurements  of  instars  3  to  28  for  males  and  females,  respectively.  Additional  points 
are  given  for  one  male  (A,  instars  29  and  30)  and  one  female  individual  (•.  instars  29  to  32).  Vertical 
and  horizontal  lines  represent  standard  errors  for  instars  24  to  28,  with  number  of  observations  given. 
For  remaining  data  points  number  of  observations  as  in  Figure  5. 


Other  relationships 

Growth  of  Crustacea  can  also  be  described  by  weight  (Hewett,  1974).  During 
this  study  weights  were  not  recorded.  Kurata  (1962)  and  Maucheline  (1977) 
pointed  out  that  an  analogous  relationship  to  weight  can  be  obtained  by  substituting 
the  cube  of  body  size  measurements  (carapace  width  or  length)  for  weight.  This 
holds  true  only  in  the  absence  of  marked  allometric  growth.  Studies  of  several 
Crustacea,  including  the  spiny  lobster  Jasus  lalandei  (Fielder,  1964)  and  the  king 
crab  Paralithodes  camtschatica  (Weber,  1967)  indicate  that,  for  practical  purposes, 
weight  varies  in  direct  proportion  to  the  cube  of  carapace  length  (y  =  ax3).  On  this 
basis  several  linear  relationships  have  been  reported. 

Kurata  (1962)  showed  a  linear  relationship  between  intermolt  period  and  the 
cube  of  body  length  for  several  Crustacea.  Regressing  D.  primitivus  intermolt 
period  (INTMT)  against  the  cube  of  carapace  width  (CW3)  resulted  in  a  linear 
relationship: 

Males:  INTMT  =  0.092  •  CW3  +  9.934  (r2  =  0.775,  N  =  260) 

Females:  INTMT  =  0.070  •  CW3  +  10.436         (r2  =  0.717,  N  =  250) 

Although  the  difference  between  slopes  was  very  small,  it  was  statistically  signif- 
icant (F  =  28.57,  P  ----  0.0001). 

In  the  common  lobster,  Homarus  vulgaris,  Hewett  (1974)  found  that  the  log 
of  growth  increment,  in  weight,  was  linearly  related  to  log  of  body  weight.  Mauche- 
line (1977)  obtained  an  analogous  relationship  for  H.  americanus  by  cubing  log 
of  increment  (log  INCR3)  and  carapace  length  (CL3)  data.  Using  the  same  ap- 
proach for  D.  primitivus  with  carapace  width,  a  significant  relationship  can  be 
obtained  for  the  first  21  instars;  for  later  instars  there  is  significant  deviation. 


220  G.  POHLE  AND  M.  TELFORD 

Males:  log  INCR3  =  0.584  •  log  C  W3  -  2.460         (r  =  0.667,  N  ==  2 1 1 ) 
Females:  log  INCR3  =  0.689  •  log  CW3  -  2.548         (r2  =  0.865,  N  ==  207) 

The  difference  in  slope  was  significant  (F  =  9.45,  P  =  0.0023). 

Hewett  (1974)  demonstrated  that  the  log  of  body  weight  was  linearly  related 
to  log  of  age.  The  analogous  relation  of  the  log  of  carapace  width  cubed  (log  CW3) 
and  log  of  age  for  D.  primitivus  was: 

Males:  log  CW3  =  2.218  •  log  AGE  --  3.158         (r2  =  0.970,  N  =  268) 
Females:  log  CW3  =  2.374- log  AGE  -  3.517         (r2  =  0.952,  N  =  260) 
The  slopes  were  significantly  different  (F  =  14.46,  P  =  0.0002). 

DISCUSSION 

In  this  study  the  highest  death  rate  occurred  during  the  short  span  of  larval 
life,  where  it  was  due  to  imperfect  molting.  In  the  wild  such  high  mortality  has 
been  observed  but  was  attributed  mostly  to  heavy  predation  (Warner,  1967).  Data 
obtained  in  the  laboratory  suggest  that  mortality  in  the  sea  may  also  be  at  least 
partially  caused  by  failure  in  molting.  Post-larval  deaths,  although  fewer,  were  also 
mostly  attributed  to  molting  difficulties  similar  to  those  described  by  Fielder  (1964) 
for  the  spiny  lobster,  Jasus  lalandei.  Fielder  noted  that  swelling  of  the  new  in- 
tegument sometimes  occurred  before  withdrawal  from  the  old  exoskeleton  was 
accomplished.  Thus,  the  time  to  complete  a  molt  is  limited.  Similarly,  either  the 
appendages  or  the  abdomen  of  D.  primitivus  could  sometimes  not  be  freed.  Without 
exception  all  individuals  in  this  study  (larvae  and  post-larvae)  molted  at  night  and 
those  which  had  not  completed  molting  by  daybreak  died. 

Changes  in  form  of  carapace  occur  in  pinnotherid  crabs  symbiotic  with  pe- 
lecypods.  Pinnotheres  ostreum  (Christensen  and  McDermott,  1958)  and  Fabia 
subquadrata  (Pearce,  1966a),  for  example,  have  hard,  flattened,  or  square  invasive 
stages,  respectively,  followed  by  more  convex  or  oval  pre-swarming  stages.  These 
differences  between  stages  are  specialized  adaptations  to  a  life  within  molluscan 
hosts.  In  both  D.  crinitichelis  (Telford,  1978a)  and  D.  primitivus,  carapace  growth 
is  isometric,  length-width  ratios  not  significantly  changing  over  the  size  range. 
Such  carapace  growth  is  typical  but  not  universal  in  crabs  (Warner,  1977).  Barnes 
(1968)  found  an  increase  in  width  over  length  during  growth  of  some  sentinel  crabs 
(Ocypodidae).  This  change  in  shape  is  a  functional  adaptation  to  side-burrowing 
(Warner,  1977).  Claims  for  various  growth  patterns  are  often  made  without  ad- 
equate statistical  testing  (Brown  and  Davies,  1972).  Re-examination  of  Barnes' 
data,  for  example,  showed  that  only  some  of  the  species  in  fact  had  linear  rela- 
tionships with  intercepts  significantly  different  from  zero  (allometry).  Finney  and 
Abele  (1981),  studying  growth  in  a  xanthid  crab  symbiotic  with  corals,  found  that 
the  carapace  of  males  and  non-ovigerous  females  increased  in  length  over  width, 
but  ovigerous  females  showed  isometric  growth.  Without  suitable  statistical  tests 
such  differences  would  not  have  been  apparent. 

Sexual  dimorphism  in  D.  primitivus  is  most  apparent  in  abdominal  growth. 
Males  and  females  can  be  distinguished  by  abdominal  widths  during  juvenile  stages 
long  before  sexual  maturity  is  reached.  The  increased  abdominal  growth  for  females 
is  necessary  in  reproduction,  where  the  abdomen  acts  in  conjunction  with  the  ster- 
num as  a  cover  to  an  incubation  chamber.  The  abdomen  of  D.  primitivus  males 
grows  more  or  less  isometrically,  whereas  females  show  positive  allometry.  In  other 
crabs  (MacKay,  1943;  Haley,  1973;  Hartnoll,  1974;  Finney  and  Abele,  1981) 


D.  PRIMITIVUS  POST-LARVAL  GROWTH  221 

female  abdominal  growth  cannot  be  explained  by  simple  allometry  with  a  single 
straight  line.  Two  phases  can  be  recognized:  the  first  of  high  positive  allometry 
(pre-puberty),  followed  by  one  of  low  positive  allometry  (post-puberty).  When 
analyzing  a  population  en  bloc,  the  change  in  allometry  is  presumptive  evidence 
of  a  pubertal  molt,  a  sudden  large  increase  in  abdominal  width  indicative  of  sexual 
maturity  (Haley,  1969;  Finney  and  Abele,  1981).  Data  of  D.  primitivus  suggest 
two  phases  leading  to  sexual  maturity  (Fig.  3).  The  first  is  a  juvenile  phase  of  low 
positive  allometric  growth,  followed  by  one  of  stronger  abdominal  growth.  It  is 
during  the  latter  phase  that  the  species  is  capable  of  maturing,  for  it  is  here  that 
pubertal  molts  for  two  females  were  observed  (Fig.  3).  Sexual  maturity  is  thus  not 
reached  at  a  constant  size  (nor  at  a  fixed  instar)  but  varies  from  individual  to 
individual.  Hence  the  observed  population  inflection  cannot  be  explained  as  a  simple 
one-step  process:  a  change  of  growth  also  occurs  before  the  pubertal  molt.  Prior 
to  successful  reproduction  various  secondary  sexual  characters  appear,  and  the 
internal  reproductive  system  must  become  functional  (Finney  and  Abele,  1981). 
The  phase  of  increased  abdominal  growth  probably  marks  one  or  more  of  these 
physiological  changes  before  the  pubertal  molt.  Haley  (1973)  observed  two  similar 
growth  phases  leading  to  sexual  maturity  in  Ocypode  ceratophthalmus.  In  that 
crab  the  second  phase  has  been  specifically  attributed  to  increased  growth  of  the 
fourth  abdominal  segment.  After  their  pubertal  molts,  growth  of  the  two  D.  prim- 
itivus female  individuals  decreased.  Post-pubertal  growth  appears  to  represent  a 
separate  phase  of  abdominal  development  but  has  not  been  fully  analyzed  due  to 
insufficient  data.  Abdominal  growth  in  Pinnotheres  pisum  (Needham,  1950)  also 
did  not  follow  simple  allometry.  Needham  fitted  the  data  onto  progressively  higher 
polynomial  functions  in  order  to  arrive  at  a  continuous  and  accurate  description 
of  growth. 

Compared  to  estimates  for  other  Crustacea  such  as  Cancer  magister  (Butler, 
1961),  the  number  of  observed  molts  for  D.  primitivus  to  reach  adult  size  seems 
high.  On  the  other  hand,  the  shrimp  Crangon  crangon  (Meixner,  1969),  had  23- 
25  post-larval  molts  before  reproduction  in  females,  and  22-25  in  males.  This  is 
similar  to  D.  primitivus,  where  pubertal  molts  occurred  after  26  and  29  instars, 
respectively. 

Growth  of  Crustacea  can  be  described  in  terms  of  duration  of  successive  in- 
termolt  periods,  which  increases  in  most  Crustacea  as  the  organism  ages  (Mauche- 
line,  1977).  There  are  some  notable  exceptions,  and  possibly  there  are  also  differ- 
ences between  sexes.  Studies  by  Reaka  (1979)  on  coral-dwelling  stomatopods  and 
Miller  el  al.  (1977)  on  marine  copepods  seem  to  indicate  a  more  or  less  constant 
molting  frequency  (isochronal  development).  In  decapod  Crustacea  a  difference 
in  intermolt  periods  of  equal-sized  males  and  females  was  found  by  Meixner  (1969) 
in  Crangon,  where  large  females  molted  more  frequently.  Large  females  of  D. 
primitivus  similarly  showed  shorter  intervals  between  molts  than  did  equal- 
sized  males. 

Duration  of  intermolt  period  is  affected  by  several  environmental  factors,  but 
especially  by  temperature  (Lasker,  1966).  Kurata  (1962)  showed  that  temperature 
variation  significantly  alters  terms  of  the  regression  equations.  In  this  study  of 
D.  primitivus,  laboratory  temperature  was  stable  and  similar  to  the  natural  envi- 
ronment. 

Changes  in  size  have  commonly  been  analyzed  in  two  ways.  The  widely  accepted 
regression  of  post-  on  pre-molt  body  size  (Hiatt,  1948),  has  recently  been  criticized 
(Maucheline,  1976,  1977)  on  theoretical  grounds  and  because  it  presupposes  con- 
stancy of  growth  increments.  Alternatively,  growth  can  be  analyzed  by  plotting 


222  G.  POHLE  AND  M.  TELFORD 

absolute  size  increase  or  percent  increase  against  body  size  (Farmer,  1973).  The 
former  usually  results  in  a  positive  relationship.  However,  the  latter  results  in  a 
negative  relationship,  percent  increments  decreasing  with  size.  In  place  of  body 
size,  successive  instar  numbers  may  also  be  used  with  similar  results. 

Decreasing  percent  increments  and  body  size  can  be  fitted  to  straight  lines 
(Maucheline,  1977)  for  many  Crustacea,  including  lobsters  (Fielder,  1964;  Thomas, 
1965))  and  crabs  (Warner,  1967;  Turoboyski,  1973).  Such  a  relationship  was  found 
for  D,  primitivus,  but  the  mean  growth  increments  were  relatively  small.  In  early 
stages  they  ranged  from  about  14  percent  to  near  zero  growth  in  later  instars. 
Results  here  are  comparable  with  such  other  pinnotherids  as  Pinnotheres  ostreum 
(10%  between  instar  1  to  2,  Sandifer,  1972),  and  Pinnixa  faba  and  P.  littoralis 
(about  20%,  Pearce,  1966b).  Larger  species,  such  as  Cancer  magister  (Butler, 
1961),  have  considerably  higher  percent  increments  (43%,  instar  1  to  2). 

Growth  increments,  however,  do  not  always  fall  on  a  single  straight  line.  For 
the  amphipod  Parathemisto  gaudichaudi  Sheader  (1981)  showed  two  distinct 
phases:  a  juvenile  phase  of  rapidly  decreasing  growth,  followed  by  a  maturing  phase 
with  more  gradually  decreasing  growth.  Ostracods  and  calanoid  copepods  are  also 
believed  to  be  exceptions  (Maucheline,  1977).  Miller  et  al.  (1977)  showed  near 
constant  percent  growth  increments  for  marine  copepods. 

Data  for  growth  rates  have  been  obtained  in  a  number  of  ways  (Burkenroad, 
1950)  including  growth  of  tagged  individuals,  change  in  size-frequency  distribution, 
and  laboratory  maintenance.  Laboratory  culture  was  chosen  in  this  study  because 
development  of  individuals  could  be  followed  for  prolonged  periods  in  controlled 
environments.  While  techniques  differ,  many  studies  have  come  to  the  conclusion 
that  growth  decreases  with  time,  irrespective  of  size  or  species  [Farmer  (1973), 
and  Hewett  (1974)  for  lobsters;  Meixner  (1969)  for  shrimps;  and  Warner  (1967), 
Weber  (1967),  and  Bennet  (1974)  for  crabs].  The  data  obtained  from  culture  of 
D.  primitivus  support  the  above  observation  and  fit  a  power  function.  This  rela- 
tionship has  been  indirectly  estimated  for  other  Crustacea  (Warner,  1967)  but 
never  demonstrated  by  continuous  long-term  culture  of  individuals.  Growth  rates 
of  D.  primitivus  are  different  for  males  and  females  (Fig.  6).  During  the  first  year 
these  differences  are  slight,  thereafter  growth  increments  for  males  decrease  (see 
equations  for  Fig.  5)  and  intermolt  periods  increase  (see  equation  for  Fig.  4).  Thus 
the  smaller  size  of  males  can  be  explained  by  a  slower  growth  rate  rather  than  by 
a  cessation  of  molting.  In  wild  populations  of  D.  primitivus  the  same  difference 
between  the  maximum  sizes  of  males  and  females  was  observed. 

Differences  in  growth  rates  between  sexes  are  known  for  other  Crustacea,  most 
of  which  have  larger  males  than  females.  In  this  respect  D.  primitivus,  as  other 
pinnotherids,  is  an  exception.  Size  differences  between  sexes  in  pinnotherids  living 
inside  molluscs  or  burrows  of  polychaetes,  such  as  Pinnotheres  ostreum  (Christen- 
sen  and  McDermott,  1958)  and  Pinnixa  cylindrica  (McDermott,  1981),  have  been 
attributed  to  differences  in  life  history.  Only  male  crabs  may  leave  for  another  host 
in  order  to  locate  additional  mates  and  thus  become  more  vulnerable  to  predation. 
This  results  in  fewer  males  reaching  a  larger  size.  In  D.  primitivus,  which  is  an 
external  parasite,  it  is  likely  that  both  male  and  female  crabs  move  from  host  to 
host,  just  as  in  D.  crinitichelis  (Telford,  1978b).  Larger  Dissodactylus  females  may 
be  necessary  for  the  production  of  enough  eggs  to  ensure  propagation  of  the  species. 

Bennet  (1974)  suggested  the  use  of  a  linear  relationship  between  percent  molt 
increment  and  premolt  weight  of  the  crab  Cancer  pagurus.  Applying  the  cubed 
carapace  length  transformation,  Maucheline  (1977)  found  this  relationship  to  be 
unsatisfactory  at  extremities  of  size  for  Homarus  americanus.  The  data  here  also 


D.  PRIMITIVUS  POST-LARVAL  GROWTH  223 

fit  only  a  small  part  of  the  size  range,  and  the  relationship  was  rejected.  However, 
a  significant  linear  relationship  was  obtained  with  these  parameters  using  a  log- 
log  transformation.  For  Homarus  vulgaris  Hewett  (1974)  obtained  a  significant 
linear  correlation  between  log  of  intermolt  period  and  the  cube  root  of  body  weight. 
As  Maucheline  (1977)  showed,  this  is  analogous  to  log  of  intermolt  period  and 
body  size  (CW  or  CL),  a  relationship  which  has  been  demonstrated  here. 

ACKNOWLEDGMENTS 

This  work  was  supported  by  the  National  Sciences  and  Engineering  Research 
Council  of  Canada  through  Operating  Grant  #  A4696.  We  express  our  thanks  to 
Miss  T.  Ortiz  and  Mrs.  J.  Caron  for  their  help  and  patience  in  the  maintenance 
of  the  animal  culture.  We  also  thank  Dr.  Finn  Sander  for  use  of  the  facilities  of 
the  Bellairs  Research  Institute  of  McGill  University,  Barbados. 

LITERATURE  CITED 

BARDACH,  J.  E.,  J.  H.  RYTHER,  AND  W.  O.  MCLARNEY.  1972.  Aquaculture.  The  farming  and  hus- 
bandry of  freshwater  and  marine  organisms.  Wiley-Interscience,  Toronto.  868  pp. 
BARNES,  R.  S.  K.  1968.  Relative  carapace  and  chela  proportions  in  some  ocypodid  crabs  (Brachyura, 

Ocypodidae).  Crustaceana  14:  131-136. 
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THE  CILIARY  JUNCTIONS  OF  SCALLOP  GILLS:  THE  EFFECTS  OF 
CYTOCHALASINS  AND  CONCANAVALIN  A 

CHARLENE  REED-MILLER1  *  AND  MICHAEL  J.  GREENBERG2 

Department  of  Biological  Sciences,  Florida  State  University.  Tallahassee,  FL  32306 

ABSTRACT 

The  ciliated  junctions  between  the  gill  filaments  of  scallop  gills  were  studied. 
Junctional  cilia  are  borne  on  both  sides  of  spurs  of  tissue — cilifers — extending  from 
the  filaments.  In  an  intact  junction,  each  cilium  is  paired  with  another  cilium  from 
a  cilifer  on  a  neighboring  filament.  An  electron  dense  band  underlies  the  plasma 
membrane  of  each  Junctional  cilium  along  the  line  of  apposition  with  its  mate. 

Cytochalasins  A,  B,  and  E  caused  gill  test  square  preparations  to  break  up  into 
their  component  filaments.  All  three  cytochalasins  disrupted  the  electron  dense 
band,  and  cytochalasins  A  and  E  also  disrupted  the  ciliary  microtubules.  These 
effects  were  reversible. 

The  paired  adhesion  of  the  Junctional  cilia  was  also  reversibly  inhibited  by 
treatment  with  Concanavalin  A  (Con  A;  100  ^g/ml).  Con  A  bound  to  the  surface 
of  the  Junctional  cilia  was  labeled  with  hemocyanin.  After  treatment  with  Con  A 
alone,  the  label  was  lightly  and  evenly  distributed  over  the  shafts  of  the  cilia,  but 
was  more  densely  concentrated  at  their  tips.  In  cytochalasin-Con  A  preparations, 
the  surface  labeling  of  the  Junctional  cilia  increased  with  the  duration  of  cyto- 
chalasin  exposure. 

INTRODUCTION 

The  gills  of  filibranchiate  bivalve  molluscs,  such  as  scallops  and  mussels  are 
composed  of  curtains  of  filaments  held  in  alignment  by  apposed  patches  of  adherent 
cilia.  These  ciliary  junctions  are  familiar  structures,  having  been  frequently  ob- 
served and  described  in  numerous  species  (Kellogg,  1890;  Rice,  1901;  Ridewood, 
1903;  Drew,  1906;  Dakin,  1908;  Outsell,  1931;  Atkins,  1937,  1938a,b;  Mattei  and 
Mattei,  1972;  and  J0rgensen,  1976).  However,  most  of  these  many  reports  were 
primarily  general  descriptions  of  filibranch  anatomy.  Moreover,  until  recently 
(Mattel  and  Mattei,  1972),  the  observations  were  made  by  light  microscopy,  so 
the  fine  structure  of  the  ciliary  junctions  remained  unknown.  This  remains  the  case, 
although  the  morphology  of  the  tip  of  a  Junctional  cilium  of  the  scallop  has  now 
been  described  in  great  detail  (Dentler,  1980). 

Ciliary  junctions  are  examples  of  a  ubiquitous  set  of  phenomena,  all  involving 
cell-cell  adhesion.  A  variety  of  systems  have  been  used  to  characterize  such  inter- 
actions, from  mating  in  yeasts  (Taylor,  1964)  and  protozoa  (e.g.,  Chalmydomonas, 
Wiese,  1969,  1974;  Snell,  1976a,b;  and  Blepharisma,  Honda  and  Miyake,  1976), 
to  the  sorting  out,  aggregation,  and  reaggregation  of  various  embryonic  tissues  and 

Received  30  March  1982;  accepted  24  May  1982. 

1  Present  address:  Department  of  Geology,  Florida  State  University,  Tallahassee,  FL  32306. 

2  Present  address:  C.   V.   Whitney  Marine  Laboratory,  University  of  Florida,  Rt.    1,  Box   121, 
St.  Augustine,  FL  32084. 

*  Author  to  whom  correspondence  should  be  addressed. 

Abbreviations:  CCA,  CCB,  CCE,  cytochalasin  A,  B,  E,  respectively;  Con  A,  concanavalin  A;  J- 
cilia,  Junctional  cilia. 

225 


226  C.  REED-MILLER  AND  M.  J.  GREENBERG 

sponge  cells  (Humphreys,  1963;  Steinberg,  1963;  Moscona,  1965).  We  thought 
that  the  ciliary  junctions  of  filibranch  gills  might  be  another  useful  system  for 
investigating  cell-cell  adhesion,  and  that  an  examination  of  their  pharmacology 
would  provide  an  efficient  first  test  of  this  possibility. 

Two  well-studied  classes  of  drugs,  the  lectins  and  the  cytochalasins,  interfere 
with  cell-cell  adhesion.  For  example,  cytochalasins  A,  B,  and  E  all  inhibit  sponge 
cell  reaggregation  (Reed,  el  al.,  1976;  Greenberg,  et  a/.,  1977),  and  they  also  block 
adhesion  of  Ehrlich  ascites  cells  to  glass  and  plastic  (Weiss,  1972).  Cytochalasin 
B  inhibits  cell  sorting  and  the  spreading  of  embryonic  chick  cells  (Steinberg  and 
Wiseman,  1 972),  and  it  also  blocks  adhesion,  aggregation,  and  spreading  of  platelets 
(Kay  and  Fudenberg,  1973). 

In  contrast  to  the  cytochalasins,  the  plant  lectin,  Concanavalin  A  (Con  A), 
interferes  with  cellular  interactions  by  agglutinating  the  participant  cells  (Kapeller 
and  Doljanski,  1972;  Sharon  and  Lis,  1972).  Some  systems  affected  by  Con  A  are 
reminiscent  of  bivalve  junctional  cilia.  For  example,  Con  A  added  to  a  culture  of 
Chlamydomonas  produces  clusters  of  cells  adhering  at  the  tips  of  their  flagella 
(see  Fig.  1,  Wiese,  1974;  and  McLean  and  Brown,  1974).  In  contrast,  Con  A  and 
another  lectin,  phytohemeagglutinin  (PHA),  inhibit  homotypic  pair  formation  in 
Blepharisma  intermedium  by  agglutinating  the  cilia  (Ricci  et  al.,  1976).  Finally, 
Con  A  tufted  and  clumped  the  cirri  on  the  ciliate,  Stylonychia  mytilus,  but  reacted 
only  weakly  with  two  other  ciliates.,  Euplotes  aediculatys  and  Tetrahymena  pyr- 
aformis  (Frisch  et  al.,  1976). 

In  this  report,  we  describe  the  ultrastructure  of  the  ciliary  junctions  of  scallop 
gills.  Then  we  examine  the  effects  on  the  ciliary  junctions  of  the  cytochalasins  and 
Con  A,  alone  and  in  combination.  Both  scanning  and  transmission  electron  micro- 
scopical observations  were  made. 

Preliminary  accounts  of  this  work  were  reported  to  the  Marine  Biological  Lab- 
oratory, Woods  Hole  (Greenberg,  1969),  and  the  American  Society  of  Zoologists 
(Reed  and  Greenberg,  1976). 

MATERIALS  AND  METHODS 

The  experimental  animals,  Argopecten  (=  Aequipecten)  irridians,  were  ob- 
tained from  the  Northeast  Marine  Specimens  Company,  Inc.,  Bourne,  Massachu- 
setts. They  arrived  in  Tallahassee,  Florida  in  good  condition,  and  were  kept  in 
aquaria,  in  filtered,  vigorously  aerated  natural  seawater  from  the  Gulf  of  Mexico 
(31  ppt),  at  15°C.  Under  these  conditions,  the  scallops  survived  for  at  least  6  days; 
but  they  were  used  for  experimentation  within  72  hours  of  their  arrival. 

Preparation  of  test  squares 

The  gills  were  dissected  from  the  animal  and  placed  in  seawater.  Small  sections 
(1  cm2),  containing  about  100  junctions,  were  cut  from  the  centers  of  the  gills. 
These  "test  squares"  remained  intact  and  active  for  about  24  hours,  propelled  about 
continuously  and  randomly  by  their  feeding  cilia. 

Test  squares  were  the  starting  material  for  all  of  the  microscopical  and  phar- 
macological observations  reported  here. 

Scanning  electron  microscopy 

Test  squares,  cut  from  the  center  of  a  gill,  were  placed  directly  in  Parducz 
fixative  (1%  OsO4:HgCl2  =:  5:1)  for  10  minutes.  The  tissue  was  then  dehydrated 


SCALLOP  GILL  CILIARY  JUNCTIONS  227 

in  a  graded  series  of  acetone  solutions.  Following  the  last  dehydration  step,  the 
material  was  dried  in  CO2  by  the  critical  point  method.  The  dried  material  was 
attached  to  SEM  stubs  with  nail  polish  and  coated  in  a  Denton  Model  502  vacuum 
evaporator  with  approximately  100  A  of  gold-palladium.  The  specimens  were  ob- 
served with  a  Cambridge  S4-10  scanning  electron  microscope  operated  at  20  KV. 

Transmission  electron  microscopy 

Test  squares  were  prepared  as  usual,  and  then  fixed  for  30  minutes  in  a  1% 
solution  of  gluteraldehyde  in  0.1  M  phosphate  buffer,  at  pH  7.5.  The  squares  were 
washed  in  the  phosphate  buffer,  and  fixation  was  then  continued  in  1%  OsO4  in 
0.1  M  phosphate  buffer  at  pH  7.5.  The  fixed  samples  were  dehydrated  in  a  graded 
series  of  alcohols,  and  then  taken  through  two  changes  of  propylene  oxide.  The 
material  was  embedded  in  Epon  812-DER  736.  Silver  to  gold  sections  were  cut 
with  a  diamond  knife  on  a  Sorvall  Porter-Blum  MT-2  ultramicrotome,  and  were 
poststained  with  uranyl  acetate  and  lead  citrate.  The  specimens  were  observed  with 
a  Philips  201  transmission  electron  microscope  operated  at  60  or  80  KV. 

Bioassay  of  cytochalasin  and  Concanavalin  A 

Test  squares  were  cut  from  the  gill  and  placed  in  10  ml  of  sea  water  in  Syracuse 
dishes,  one  section  per  dish.  Stock  solutions  (2  mg/ml)  of  cytochalasins  A,  B,  and 
E  (Imperial  Chemicals  Industries,  Ltd.,  Macclesfield,  Cheshire,  U.  K.)  in  dimethyl- 
sulfoxide  (DMSO)  were  prepared,  and  aliquots  taken  up  in  a  microliter  syringe 
were  transferred  to  the  sea  water  in  the  Syracuse  dishes  to  achieve  the  appropriate 
test  concentration  (i.e.,  5,  6,  7,  8,  9,  or  10  Mg/ml).  The  time  between  the  application 
of  a  dose  of  cytochalasin,  and  the  complete  dissociation  of  a  test  square  into  its 
component  filaments,  was  taken  as  a  measure  of  the  effect  of  that  dose. 

Concanavalin  A  (Con  A;  Miles-  Yeda  Ltd.,  Miles  Laboratories,  Kankakee,  IL) 
activity  was  assayed  in  an  identical  manner  to  that  described  for  the  cytochalasins, 
except  that  the  drug  was  dissolved  in  glass-distilled  water.  The  test  concentrations 
of  Con  A  were  25,  50,  75,  and  100 


Electron  microscopic  assessment  of  drug  actions 

Test  squares  of  the  gill  were  incubated  in  the  series  of  cytochalasin  test  con- 
centrations in  seawater.  After  5,  10,  or  15  minutes  of  incubation,  the  tissues  were 
prepared  for  scanning  (SEM)  or  transmission  electron  microscopy  (TEM),  as  de- 
scribed above. 

Similarly,  test  squares  were  incubated  for  10  minutes  in  the  Con  A  test  solutions, 
rinsed  in  filtered  sea  water,  and  then  fixed  for  SEM. 

In  order  to  rule  out  any  fixation  artifacts  that  could  be  interpreted  as  drug- 
induced  effects,  control  (untreated)  tissue  was  fixed  for  electron  microscopy  with 
every  group  of  experimental  tissue. 

Incubation  in  cytochalasins  followed  by  incubation  in  Con  A 

Stock  solutions  (2  mg/ml)  of  the  cytochalasins  were  taken  up  in  a  microliter 
syringe  and  transferred  to  10  ml  of  sea  water;  the  final  concentration  was  10  Mg/ 
ml.  After  10  minutes  in  the  cytochalasin  solution,  the  gills  were  rinsed  in  sea  water 
and  100  ng/m\  Con  A  was  added  immediately.  Following  this  second  incubation 
(10  min)  in  Con  A,  the  gills  were  rinsed  and  fixed  for  SEM. 


228  C.  REED-MILLER  AND  M.  J.  GREENBERG 

Hemocyanin  labeling  procedure 

The  test  squares  were  fixed  in  Parducz  solution,  rinsed,  incubated  in  100 
ml  Con  A  for  10  minutes,  then  rinsed  again.  This  was  followed  by  an  incubation 
in  1  mg/ml  hemocyanin  (keyhole  limpet;  lyophilized  powder,  ammonium  sulfate 
free;  Calbiochem,  La  Jolla,  CA).  The  tissue  was  then  dehydrated  and  prepared  for 
SEM  as  usual. 

Since  the  amount  and  distribution  of  Con  A  binding  depends  on  whether  the 
tissue  is  fixed  before  or  after  exposure  to  the  lectin,  other  test  squares  were  prepared 
as  described  above,  except  that  the  incubations  with  Con  A  and  hemocyanin  pre- 
ceded fixation  in  Parducz  solution. 

Controls  for  the  hemocyanin  labeling  procedure  were  carried  out  with  the  hapten 
inhibitor  of  Con  A  binding,  alpha-D-methylmannoside  (Brown  and  Revel,  1976). 
The  gill  test  squares  were  incubated  for  10  minutes  in  0.1  M  alpha-D-methylman- 
noside, either  with,  or  after,  the  Con  A  incubation,  but  before  hemocyanin  labeling. 

The  effects  of  cytochalasin  incubation  time  on  the  number  and  arrangement 
of  Con  A  receptors  on  the  J-cilia  were  assessed  by  the  following  procedure.  A  set 
of  test  squares  were  incubated  in  10  ^1/ml  cytochalasin.  Sample  squares  were 
removed  from  the  medium  at  1 -minute  intervals,  from  0  to  30  min.  As  the  tissues 
were  removed  from  the  cytochalasin  solution,  they  were  fixed,  incubated  for  10 
minutes  in  100  Mg/ml  Con  A,  rinsed,  incubated  for  10  minutes  in  1  mg/ml  he- 
mocyanin, rinsed  again,  and  prepared  for  SEM  as  usual. 

Control  experiments  to  test  the  effects  of  drug  solvents  were  run,  but  no  effects 
were  observed.  The  various  drug  actions  described  below  were  consistently  observed 
on  the  junctional  cilia,  whereas  the  appearance  of  the  rest  of  the  gill  tissue  was  the 
same,  whether  or  not  it  was  treated.  Therefore,  osmotic  damage  to  the  membranes 
during  fixation,  even  if  it  occurred,  could  not  have  contributed  to  the  results. 

RESULTS 

The  gills  of  scallops  are  parallel  linear  arrays  of  W-shaped  filaments,  suspended 
from  their  centers  by  the  gill  axis.  The  gills  are  plicate,  or  pleated,  with  the  plicae 
(the  pleats)  occurring  about  every  eighth  to  fourteenth  filament.  A  large  filament, 
the  principal  filament,  is  located  at  the  apex  of  each  plica  (Fig.  1). 

There  are  two  types  of  cilia  on  the  gill — the  feeding  cilia,  distributed  along  the 
length  of  the  filament;  and  the  junctional  cilia,  located  only  on  tongue-like  pro- 
jections from  the  filaments  (Fig.  2).  The  feeding  cilia  create  the  feeding  and 
respiratory  water  current,  and  sort  and  distribute  the  small  particles  borne  on  this 
incurrent  stream.  The  feeding  cilia  are  about  10  /urn  long. 

In  scallop  gills,  the  tongue-like  projections  bearing  the  junctional  cilia  (or  J- 
cilia)  occur  at  intervals  of  0. 1  mm  along  the  length  of  the  filaments.  The  projections 
on  adjacent  filaments  are  in  register,  and  overlap  like  a  set  of  spoons  (Fig.  1).  We 
call  these  tongues  ci lifers  (i.e.,  "cilia-bearers").  The  cilifers  assume  a  variety  of 
shapes  ranging  from  round  to  elongate.  The  regular  filaments  have  one  cilifer  per 
0.1  mm  of  length,  and  all  of  the  cilifers  point  in  the  same  direction.  In  contrast, 
the  principals  have  two  oppositely  directed  (anterior  and  posterior)  cilifers  occurring 

FIGURE  1 .  Scallop  gill  filaments  with  two  rows  of  ciliary  junctions.  The  cilifers  (structures  bearing 
the  junctional  cilia)  are  in  rows  perpendicular  to  the  filaments.  Note  the  plicated  appearance  of  the  gill, 
pf  =  principal  filament;  c  =  cilifer.  Bar  =  50  nm. 

FIGURE  2.  One  of  the  rows  of  ciliary  junctions  oriented  at  right  angles  to  the  gill  filaments.  The 
junctional  cilia  (J)  are  visible  between  the  intact  junctions  and  on  the  single  unpaired  cilifer.  The  feeding 
cilia  (F)  are  along  the  length  of  the  filaments.  Bar  =  40  ^m. 


SCALLOP  GILL  CILIARY  JUNCTIONS 


229 


FIGURE  3.  An  intact  ciliary  junction.  Each  J-cilium  is  paired  along  its  length  with  one  from  the 
opposite  side  of  the  junction.  The  tip  of  each  cilium  of  a  pair  is  hooked  around  the  base  of  its  mate 
(arrows).  Bar  =  1  ^m. 

FIGURE  4.  Higher  magnification  of  an  area  in  Figure  3,  showing  the  tip  of  one  junctional  cilium 
hooked  around  the  base  of  its  mate  (arrow).  Bar  =  250  nm. 


230  C.  REED-MILLER  AND  M.  J.  GREENBERG 

at  0.1  mm  intervals  (Fig.  1).  Junctional  cilia  cover  both  sides  of  the  cilifers  on  the 
regular  filaments,  but  only  one  side  of  those  located  on  the  principal  filaments.  The 
J-cilia  (6  ^m  long  on  the  average)  are  shorter  than  the  feeding  cilia.  There  are 
about  800  cilia  on  each  side  of  a  cilifer,  so  the  density  is  about  6.7  X  103/rnm2. 

The  surfaces  of  both  the  feeding  and  J-cilia  are  rough  and  wrinkled.  Each  J- 
cilium  is  closely  apposed  along  its  entire  length  to  one  other  cilium  from  a  cilifer 
on  a  neighboring  filament  (Fig.  3).  Therefore,  the  cilia  on  the  facing  surfaces  of 
the  two  cilifers  making  up  one  junction  interdigitate.  Moreover,  the  last  0.4  /j.m 
of  the  tip  of  each  of  these  paired  cilia  is  hooked  around  the  base  of  its  mate  (Fig. 
4).  The  pairing  of  the  J-cilia  is  evident  in  TEM  cross  sections  (Fig.  5).  In  such 
sections,  the  arms  of  the  microtubules  in  each  cilium  of  a  pair  are  seen  to  be 
oriented  in  opposition  to  those  of  its  mate,  confirming  that  the  two  cilia  arise  from 
opposite  cilifers.  In  addition,  an  electron  dense  band  underlies  the  plasma  mem- 
branes of  adhering  J-cilia  along  the  line  of  their  apposition  (Fig.  5). 

Dissociation  of  test  squares  by  cytochalasin 

A  sufficient  dose  of  any  of  the  three  cytochalasins,  A,  B,  and  E,  caused  test 
square  preparations  of  scallop  gills  to  dissociate  into  their  component  filaments. 
The  uncoupled  filaments  swam  around  the  dish  propelled  by  their  feeding  cilia; 
they  collided,  but  never  stuck,  even  when  two  cilifers  made  contact.  If  the  cyto- 
chalasins were  removed  by  replacement  of  the  solution  with  normal  medium,  then 
cilifers  making  random  contact  would  adhere,  and  mats  of  filaments  would  occur. 

The  dissociation  of  the  test  squares  was  dependent  on  three  factors:  the  dose 
of  cytochalasin  used;  the  time  that  the  cytochalasin  was  left  in  contact  with  the 
preparation;  and  the  cytochalasin  being  tested.  At  any  dose,  cytochalasin  E  was 
more  potent  than  cytochalasins  A  or  B;  the  latter  were  equiactive  (Table  I). 

Ultrastructural  effects  of  the  cytochalasins 

The  J-cilia  on  the  surface  of  mechanically  isolated,  but  otherwise  untreated, 
cilifers  are  uniformly  unpatterned  (Fig.  6,  control).  Cytochalasin  treatment  changed 
this  picture.  The  first  noticeable  effect  was  the  loss  of  randomness,  and  the  formation 
of  tufts  of  from  10-20  cilia  (Fig.  7).  Within  each  tuft,  the  cilia  were  joined  only 
along  their  shafts;  the  tips  were  free  and  were  often  hooked  over,  or  even  curled 
into  small  knots.  When  the  dose  of  cytochalasin  was  high,  or  the  incubation  time 
was  long,  a  swelling  or  blebbing  of  the  cilium  appeared  just  below  the  tip.  In  some 
cases,  and  always  at  low  doses  of  cytochalasin,  the  material  was  fixed  before  the 
test  squares  had  completely  dissociated  into  their  constituent  filaments;  yet  tufting 
at  these  intact  junctions  was  already  occurring  (Fig.  8). 

TABLE  I 

The  rate  of  scallop  gill  dissociation  produced  by  the  cytochalasins  increases  with  dose. 


Dose  (^g/ml) 

Cytochalasin 

5 

6 

7 

8 

9 

10 

A 

31.5 

26 

24.5 

21 

18 

14 

B 

31 

31.5 

24 

20 

13.5 

8.5 

E 

24.5 

18 

15.5 

14 

11 

6.5 

Five  sections  of  scallop  gill  (1  cm2)  were  tested  at  each  dose;  the  mean  time  (min)  required  for 
dissociation  into  individual  filaments  is  tabulated. 


SCALLOP  GILL  CILIARY  JUNCTIONS 


231 


r\  ***    ^*  ^    54 

^00;.,0« 


f 


FIGURE  5.  A  transmission  electron  micrograph  of  a  cross  section  through  an  intact  junction, 
showing  the  pairing  of  the  J-cilia  and  the  electron-dense  band  underlying  the  ciliary  membrane  (arrow). 
Bar  =  500  nm. 

FIGURE  6.  An  untreated,  control  cilifer  showing  the  unpatterned  arrangement  of  the  junctional 
cilia  over  the  surface.  Bar  =  10  ^m. 

FIGURE  1 .  A  cilifer  after  exposure  to  10  Mg/ml  cytochalasin  E  for  one  hour.  Note  the  tufting  of 
the  J-cilia.  Compare  with  Figure  6.  Bar  =  15  ^m. 

FIGURE  8.  A  ciliary  junction  following  treatment  with  10  Mg/ml  cytochalasin  A.  The  two  cilifers 
making  up  the  junction  are  attached  by  tufted  junctional  cilia.  Bar  =  1  nm. 


Transmission  electron  microscopy  showed  two  additional  effects  of  the  cyto- 
chalasins  on  the  J-cilia:  the  electron-dense  band  underlying  the  membrane  of  each 
J-cilium,  on  the  side  apposed  to  its  mate,  disappeared  (Fig.  9);  and  the  microtubules 
were  poorly  defined,  and  almost  muddy  in  appearance  following  the  administra- 
tion of  either  cytochalasin  A  or  E  (Fig.  10).  The  disruption  of  the  microtubules 
occurred  only  at  relatively  high  doses  of  cytochalasin  A  or  E  (about  15  /ig/ml), 


232 


C.  REED-MILLER  AND  M.  J.  GREENBERG 


FIGURE  9.  A  transmission  electron  micrograph  of  J-cilia  following  treatment  with  15  ng/m\  cy- 
tochalasin  B.  The  microtubules  of  the  unpaired  J-cilia  are  well  defined,  but  the  electron  dense  band  is 
absent.  Bar  =  500  ^m. 

FIGURE  10.     A  transmission  electron  micrograph  of  J-cilia  following  treatment  with   15 
cytochalasin  E.  The  tubules  and  their  arms  appear  muddy.  Bar  =  250  nm. 


SCALLOP  GILL  CILIARY  JUNCTIONS  233 

whereas  lower  doses  (about  5  yug/mO  were  sufficient  to  effect  the  disappearance 
of  the  electron-dense  band.  The  microtubules  were  not  affected  by  any  dose  of 
cytochalasin  B. 

In  contrast  to  the  J-cilia,  the  feeding  cilia  were  apparently  unaffected  by  the 
cytochalasins,  even  after  large  doses. 

Effects  of  Concanavalin  A 


Treatment  with  100  Mg/ml  of  Con  A  for  10  minutes  usually  caused  test  squares 
to  separate  into  their  component  filaments.  But  in  some  tests,  the  dissociation  was 
not  complete,  and  some  ciliary  junctions  remained  coupled.  The  major  effect  of 
Con  A  on  the  junctional  cilia  was  the  production  of  tufts  of  from  10-20  cilia  over 
the  entire  face  of  the  cilifers  (Fig.  11).  But  these  tufts,  in  contrast  with  those 
produced  by  the  cytochalasins,  were  clumped  only  at  their  tips;  the  shafts  of  the 
cilia  were  free  from  contact  with  their  neighbors.  Frequently  the  tips  (1.5-2.0  ^m) 
of  the  J-cilia  were  swollen  3-4-fold  by  treatment  with  Con  A  (Fig.  12).  The  same 
morphological  changes  were  found  following  exposure  to  25,  50,  or  75  /ig/ml  Con 
A  for  10  minutes. 

Combined  effects  of  Con  A  and  the  cytochalasins 

The  10-minute  preincubation  with  10  ng/m\  cytochalasin  caused  the  test 
squares  to  disperse  into  individual  filaments.  When  these  filaments  were  rinsed  and 
treated  for  10  minutes  with  Con  A,  the  J-cilia  again  tufted  into  groups  of  from 
10-20  cilia  (Fig.  13).  But  instead  of  clumping  only  at  their  tips  as  with  Con  A 
alone,  or  clumping  only  along  their  shafts  as  with  cytochalasin  alone,  the  J-cilia 
were  very  closely  apposed  to  one  another  along  their  entire  lengths. 

Distribution  of  Con  A  binding  sites 

If  gills  were  first  fixed  for  SEM,  and  then  exposed  to  Con  A  and  hemocyanin, 
label  was  evenly  distributed  over  the  shafts  of  the  unpaired  J-cilia  (Fig.  14).  Those 
J-cilia  that  remained  coupled  to  their  mates  on  opposing  cilifers  were  virtually 
unlabeled  (Fig.  15).  Similarly,  only  a  small  amount  of  hemocyanin  was  distributed 
over  the  feeding  cilia  and  the  rest  of  the  gill  tissue,  probably  labeling  some  mu- 
copolysaccharide  that  had  survived  the  fixation. 

If  the  gills  were  treated  with  Con  A  for  10  minutes  before  fixation  and  labeling, 
the  distribution  of  label  was  the  same,  but  the  density  was  lower.  Again,  there  was 
little  label  on  those  J-cilia  that  were  still  paired  (Fig.  16);  but  it  was  greater  than 
on  coupled  preparations  that  had  been  fixed  before  being  exposed  to  Con  A  and 
hemocyanin. 

FIGURE  11.  A  cilifer  after  treatment  with  Con  A  showing  the  tufting  of  the  J-cilia  over  the  surface 
of  the  cilifer.  Compare  with  Figures  6  and  7.  Bar  =  10  /^m. 

FIGURE  12.  Tufted  cilia  on  the  outside  edge  of  a  cilifer  after  treatment  with  Con  A.  The  cilia  do 
not  make  contact  at  their  shafts;  the  only  area  of  union  is  at  their  tips.  Bar  =  1  /urn. 

FIGURE  13.  A  cilifer  after  treatment  with  10  Mg/ml  cytochalasin  E  for  one  hour  followed  by  100 
Mg/ml  Con  A  for  10  minutes,  showing  the  tufting  of  the  cilia.  Compare  with  Figures  6,  7  and  1  1.  Bar 
=  10  Mm 

FIGURE  14.  The  distribution  of  hemocyanin  label  on  the  shafts  of  J-cilia  (arrows).  The  tissue  was 
fixed,  then  incubated  with  Con  A  and  hemocyanin.  Bar  =  250  nm. 

FIGURE  15.  A  junction  after  fixation  and  treatment  with  Con  A  and  hemocyanin.  One  ciliary  pair 
has  separated,  and  hemocyanin  label  is  present  on  the  tips  of  the  separated  cilia  (arrows).  Bar  =  1  ^m. 


234 


C.  REED-MILLER  AND  M.  J.  GREENBERG 


FIGURE  16.  A  partially  intact  junction  fixed  after  a  10-minute  exposure  to  cytochalasin.  The 
partially  coupled  J-cilia  have  some  label;  underneath  them  are  J-cilia  which  remain  paired  and  unlabeled 
(arrows).  Bar  =  1  ^m. 

FIGURE  17.  Junctional  cilia  fixed  and  simultaneously  treated  with  cytochalasin,  and  then  labeled. 
There  is  no  label  on  the  surface  of  the  cilia.  Bar  =  1  fim. 


SCALLOP  GILL  CILIARY  JUNCTIONS  235 

In  summary,  J-cilia  fixed  before  exposure  to  Con  A  and  hemocyanin  had  less 
label  than  those  fixed  after  labeling.  In  either  case,  there  was  very  little  labeling 
on  paired  J-cilia. 

The  effect  of  the  cytochalasins  on  the  distribution  of  Con  A  binding  sites 

The  density  of  labeled  Con  A  binding  sites  on  the  J-cilia  varied  directly  with 
the  duration  of  exposure  to  the  cytochalasins  before  fixation.  When  cytochalasin 
was  added  with  the  fixative  (0  time),  no  label  bound  to  the  J-cilia  (Fig.  17); 
moreover,  there  was  no  discernable  increase  in  labeling  at  incubation  times  of  up 
to  4  minutes.  But  if  the  tissue  was  preincubated  with  cytochalasin  for  5  minutes, 
the  amount  of  label  increased  noticeably  from  that  observed  at  0  time  (Fig.  18), 
and  the  hemocyanin  marker  was  widely  dispersed  over  the  entire  ciliary  surface. 
The  label  was  denser  after  10  minutes  of  exposure  to  cytochalasin;  after  1 5  minutes, 
almost  the  entire  ciliary  surface  was  obscured  by  the  heavy  hemocyanin  label  (Fig. 
19).  Longer  incubation  times  would  not  increase  this  response.  No  label  was  found 
anywhere  on  those  J-cilia  that  remained  paired  (Fig.  20). 

As  a  control  for  the  cytochalasin-Con  A  experiments,  gills  were  exposed  to  cy- 
tochalasin, followed  by  incubation  with  Con  A  and  alpha-D-methylmannoside,  a 
hapten  inhibitor  for  Con  A.  Following  this  treatment,  there  was  little,  if  any  label 
present  on  the  J-cilia,  and  they  did  not  tuft  or  clump  (Fig.  21).  If  the  gills  were 
incubated  only  with  alpha-D-methylmannoside  and  Con  A,  the  J-cilia  tufted  and 
were  united  at  their  tips  (Fig.  22). 

DISCUSSION 

Three  salient  features  emerge  from  our  observations  of  the  ultrastructure  of 
Argopecten  ciliary  junctions.  First,  only  the  cilia  of  apposed  cilifers  are  adherent; 
the  cilia  on  isolated  cilifers  do  not  adhere.  Second,  the  cilia  adhere  in  pairs.  Third, 
the  membranes  of  adherent  paired  cilia  (but  not  of  detached  J-cilia)  are  modified 
along  the  line  of  their  apposition  into  thickened,  electron-dense  bands.  These  char- 
acteristics, particularly  the  narrow  electron-dense  band,  have  also  been  observed 
in  the  ciliary  junctions  of  Mytilus  perna  (Mattei  and  Mattel,  1972),  and  they  are 
probably  common  to  ciliary  junctions  throughout  the  Bivalvia. 

The  characteristic  pairing  of  adherent  cilia,  and  their  membrane  modification, 
precludes  the  possibility  that  the  adhesion  could  be  due  simply  to  frictional  resis- 
tance between  the  tightly  interdigitated  sets  of  apposed  cilia.  Rather,  these  struc- 
tural features  suggest  that  the  mechanisms  of  adhesion  must  include  some  specific 
molecular  interaction  between  J-cilia.  This  conclusion  is  supported  by  two  obser- 
vations of  Murakami  (1962,  1963):  that  the  J-cilia,  whether  dissociated  or  paired, 
are  not  stiff  (e.g.,  like  hair  brush  bristles),  but  are  flexible  and  motile;  and  that  the 
connecting  force  of  the  junction  is  dependent  on  the  ionic  composition  of  the  me- 

FIGURE  18.  Part  of  a  cilifer  fixed  5  minutes  after  the  addition  of  cytochalasin,  then  labeled.  There 
is  some  label  on  the  cilia.  Bar  =  1  ^m. 

FIGURE  1 9.  J-cilia  fixed  after  1 5  minutes  of  exposure  to  cytochalasin,  then  labeled.  There  is  labeling 
all  over  the  ciliary  surface.  Compare  with  Figures  17  and  18.  Bar  =  1  ^m. 

FIGURE  20.  A  junction  treated  for  10  minutes  with  cytochalasin,  then  fixed  and  labeled  with  Con 
A  -  hemocyanin.  There  is  little  label  on  those  J-cilia  which  remain  paired,  but  the  uncoupled  J-cilia  are 
heavily  labeled.  Bar  =  1  urn. 

FIGURE  21.  J-cilia  exposed  to  cytochalasin  for  15  mintues,  then  incubated  with  Con  A  and  alpha- 
methylmannoside  and  labeled  with  hemocyanin.  There  is  little,  if  any  label  on  the  cilia.  Bar  =  1  nm. 

FIGURE  22.  J-cilia  after  incubation  in  alpha-methylmannoside  and  Con  A  (10  min).  The  cilia  are 
tufted  and  adhere  at  their  tips.  Bar  =  1  Mm. 


236  C.  REED-MILLER  AND  M.  J.  GREENBERG 

dium.  The  disruption  of  ciliary  junctions  by  the  cytochalasins  and  Con  A  also 
suggest  a  molecular,  rather  than  a  mechanical,  adhesion. 

The  above  argument  notwithstanding,  a  few  additional  observations  imply  that 
the  adhesion  between  J-cilia  has  a  mechanical  component  as  well  as  a  chemical 
one.  First,  the  mutual  hooking  of  each  ciliary  tip  around  the  adjacent  comple- 
mentary ciliary  base  could  be  providing  a  mechanical  link.  Second,  the  basis  for 
such  a  linkage  is  suggested  by  a  recent  ultrastructural  study  of  ciliary  tips,  including 
those  of  the  J-  and  feeding  cilia  of  Argopecten  gills  (Dentler,  1980).  The  J-cilia 
tips  are  unique  in  that  their  central  microtubule  caps  are  connected  to  the  outer 
tubule  doublets  by  the  distal  filaments.  This  arrangement  results  in  a  direct  me- 
chanical connection  between  the  distal-most  patch  of  ciliary  membrane  (attached 
to  the  central  microtubule  cap)  and  the  outer  doublet.  Given  this  connection,  to- 
gether with  the  structural  discontinuity  in  the  cilium  at  the  ends  of  the  outer 
doublets,  differential  shortening  of  the  doublets  (Satir,  1968)  might  well  cause  the 
tip  of  the  cilium  to  hook  over.  Third,  low  doses  of  cytochalasins  sometimes  produce 
tufting  while  the  junction  is  still  intact;  and  in  such  instances,  the  tips  of  the  paired 
cilia  remain  wrapped  around  their  mates,  but  away  from  the  base,  i.e.,  towards  the 
middle  of  the  shaft.  Thus,  the  proposed  mechanical  link  seems  to  slip  under  stress 
and  to  be  separable  from  the  adhesion  occurring  along  the  paired  shafts.  Finally, 
Murakami  (1962)  showed  that  when  previously  separated  cilifers  of  Mytilus  were 
held  together,  they  would  adhere  after  "several  minutes,"  but  the  force  of  adhesion 
would  increase  to  a  maximum  by  about  10  hours.  This  observation,  which  we  have 
repeated  qualitatively  on  scallop  gills,  is  suggestive  of  at  least  two  adhesive  pro- 
cesses, one  occurring  rapidly,  and  one  developing  more  slowly.  We  cannot,  at  pres- 
ent, identify  the  components,  and  other  interpretations  of  the  data  are  of  course 
possible;  yet  we  conclude  that  a  mechanical  contribution  to  adhesion  is  a  reasonable 
possibility  that  should  remain  open. 

The  ciliary  junctions  of  bivalve  filibranch  gills  are  reminiscent  of  heterctypic 
interactions  between  cilia  of  Protozoa.  For  example,  the  early  stage  of  conjugation 
in  the  ciliate,  Blepharisma  intermedium,  is  characterized  by  a  ciliary  union  between 
two  cells,  one  from  mating  type  I,  and  another  from  mating  type  II  (Honda  and 
Miyake,  1976).  Again,  conjugation  in  Chlamyodomonas  is  initiated  by  contact 
between  the  tips  of  flagella  of  sexually  different  gametes  (Wiese,  1969,  1974). 

Homotypic  ciliary  complexes  are  also  seen  in  the  Protozoa,  particularly  in  the 
adoral  zone  of  membranelles,  the  undulating  membranes,  and  the  cirri  of  ciliates. 
The  cilia  in  these  complex  structures  are  closely  apposed,  like  those  of  the  bivalve 
ciliary  junction,  but  they  are  not  paired;  and  the  membrane  modifications  include 
numerous  small  projections  along  the  shafts  (Roth,  1956;  Randall  and  Jackson, 
1958;  Giese,  1973),  and  parallel  rows  of  intramembrane  particles  at  the  tips  (Mon- 
tesano  el  al.,  1981).  Such  complexes  are  most  similar  to  the  latero-frontal  cirri, 
feeding  organelles  of  mussel  and  oyster  gills  (Owen,  1974;  Owen  and  McCrae, 
1976).  Finally,  the  adhesion  between  the  flagellum  and  the  body  wall  of  a  try- 
panosome  (the  "undulating  membrane")  is  mechanically  dissociable  and  seems  to 
involve  membrane  modifications  (reviewed  by  Hoare,  1972).  This  system  is  thus 
reminiscent  of  J-cilia  adhesion. 

Effects  of  the  cytochalasins 

Four  ultrastructural  changes  appeared  following  the  application  of  the  cyto- 
chalasins. First,  all  three  of  the  compounds  tested — cytochalasins  A,  B,  and  E— 
eliminated  the  electron-dense  bands  in  the  apposed  membranes  of  paired  cilia.  One 


SCALLOP  GILL  CILIARY  JUNCTIONS  237 

of  the  classical  mechanisms  of  action  of  the  cytochalasins  is  the  depolymerization 
of  actin-like  microfilaments  (Wessels  et  al.,  1971).  For  example,  such  filaments 
found  in  BALE  and  3T3  cells  disappear  after  cytochalasin  treatment  (Gershenbaum 
et  al.,  1974),  and  the  microfilament-dependent  locomotion  of  glial  cells  stops  after 
exposure  to  cytochalasin  B  (Spooner  et  al.,  1971).  Thus,  the  disappearance  of  the 
electron-dense  band  implies  that  this  structure  is  composed  of  cytochalasin-sensitive 
microfilaments.  If  this  were  the  case,  then  the  bands  might  be  conceived  of  as  strips 
of  microfilaments  holding  in  alignment  specialized  membrane  receptors  responsible 
for  the  pairing  of  J-cilia.  Such  receptor-microfilament  associations  have  previously 
been  described  (Brown  and  Revel,  1976).  The  problem  with  this  hypothesis  is  that 
the  electron-dense  band  disappears  even  if  the  ciliary  junction  is  merely  pulled 
apart.  Thus,  the  possibility  remains  that,  although  the  cytochalasins  dissociate 
junctions,  the  disappearance  of  the  band  could  be  an  indirect  consequence  of  that 
dissociation. 

The  second,  more  gross,  effect  of  all  of  the  cytochalasins  was  the  formation  of 
tufts  of  ten  to  twenty  J-cilia  on  the  separated  cilifers.  Tufting  may  represent  an 
increase  in  homotypic,  as  compared  with  heterotypic,  adhesiveness  leading  to  the 
breakage  of  the  bonding  between  pairs  of  J-cilia.  Since  we  have  seen  tufts  on 
dissociating,  but  stll  paired,  cilifers,  this  remains  a  reasonable  notion.  However,  the 
reverse  possibility,  that  tufting  is  a  consequence  of  dissociation  or  the  disappearance 
of  the  electron-dense  band,  and  only  indirectly  caused  by  cytochalasin,  is  not  prob- 
able. That  is,  mechanical  dissociation  leads  to  the  loss  of  the  electron-dense  band, 
but  not  to  tufting.  In  fact,  Murakami  (1963)  showed  that  the  J-cilia  become  vig- 
orously active  when  the  ciliary  junction  is  pulled  apart. 

The  third  effect  of  the  cytochalasins,  an  increase  in  the  number  of  hemocyanin- 
labeled  Con  A  binding  sites  on  the  J-cilia,  could  come  about  in  two  ways.  Cyto- 
chalasins could  stimulate  the  production  of  new  Con  A  binding  sites,  or  they  could 
modify  the  membrane,  making  extant,  but  unavailable,  binding  sites  accessible  to 
Con  A.  The  disappearance  of  the  electron-dense  band  could  reflect  a  modification 
of  the  J-cilia,  which  might  also  expose  or  reactivate  previously  masked  Con  A 
receptors.  The  cytochalasins  are  known  to  affect  the  distribution  and  number  of 
cell  surface  Con  A  receptors  (binding  sites)  (Ash  and  Singer,  1976;  Nicholson  and 
Poste,  1976;  Schlessenger  et  al.,  1976;  Brown  and  Revel,  1976). 

A  fourth  effect  of  the  cytochalasins  was  the  disruption  of  the  microtubules  in 
the  J-cilia.  However,  only  cytochalasins  A  and  E  had  this  effect,  and  only  at 
relatively  high  concentration  (15  Mg/ml).  Cytochalasin  A  has  been  shown  to  bind 
to  tubulin  and,  in  fact,  to  compete  with  colchicine  for  a  binding  site  (Himes  and 
Houston,  1976).  However,  the  ciliary  junction  is  disrupted  by  low  doses  of  CCB 
(cytochalasin  B)  with  no  apparent  effect  on  the  microtubules  in  the  J-cilia.  We 
therefore  conclude  that  the  actions  of  CCA  and  CCE  on  microtubules  are  irrelevant 
to  the  adhesive  mechanism  responsible  for  the  ciliary  junctions. 

Effects  of  concanavalin  A 

Like  the  cytochalasins,  Con  A  dissociates  ciliary  junctions  and  causes  tufting 
of  the  J-cilia  on  the  isolated  cilifers.  However,  Con  A-tufted  cilia  adhere  at  their 
tips,  as  do  protozoan  cilia  or  flagella  after  treatment  with  this  lectin  (see  references 
in  Introduction). 

Presumably,  Con  A  dissociates  ciliary  junctions  by  binding  to  receptors  on  the 
J-cilia  membranes,  thereby  interfering  with  the  molecular  interaction  between  ap- 
posed  ciliary  pairs.  We  suggest  tentatively,  that  the  Con  A  receptors  may  be  con- 


238  C.  REED-MILLER  AND  M.  J.  GREENBERG 

centrated  along  the  electron-dense  band,  held  in  this  array  by  the  presumed  mi- 
crofilamentous  cytoskeleton  of  the  band.  Such  an  arrangement  would  explain  the 
effectiveness  of  Con  A  in  dissociating  J-cilia.  It  might  also  explain,  in  part,  the 
increase  in  Con  A  binding  site  labeling  by  cytochalasin,  concomitant  with  the 
disappearance  of  the  electron-dense  band. 

ACKNOWLEDGMENTS 

The  authors  thank  William  I.  Miller,  III  for  assistance  with  the  scanning  elec- 
tron microscopy.  We  are  grateful  to  Mr.  Dennis  Cassidy  and  Mr.  Tom  Fellers  of 
the  Antarctic  Research  Facility  at  Florida  State  University  who  helped  with  some 
of  the  plate  photography.  This  is  Contribution  Number  94  from  the  Tallahassee, 
Sopchoppy  and  Gulf  Coast  Marine  Biological  Association.  Supported  by  NIH  grant 
HL-09283. 

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BIOCHEMICAL  CHARACTERISTICS  OF  MACROURID  FISHES 
DIFFERING  IN  THEIR  DEPTHS  OF  DISTRIBUTION 

JOSEPH  F.  SIEBENALLER,1 '  GEORGE  N.  SOMERO,2'  AND  RICHARD  L.  HAEDRICH3' 

1  School  of  Oceanography.  Oregon  State  University,  Marine  Science  Center.  Newport.  OR  97365; 
2 Marine  Biology  Research  Division.  Scripps  Institute  of  Oceanography,  University  of  California- 
San  Diego,  La  Jolta.  CA  92093;  Department  of  Biology,  Memorial  University  of  Newfoundland, 
St.  John's,  Newfoundland  AlB  3X9,  Canada;  and,  *  Marine  Biological  Laboratory, 

Woods  Hole,  MA  02543 

ABSTRACT 

Enzymic  activities  (units  per  gram  wet  weight  of  tissue)  were  measured  in  white 
skeletal  muscle  and  brain  tissue  of  five  species  of  macrourid  (rattail)  fishes  occurring 
over  an  approximately  5000  m  depth  gradient.  Muscle  protein  and  water  contents 
were  also  determined.  All  species  exhibited  extremely  low  amounts  of  muscle  en- 
zymic  activity  for  the  glycolytic  enzymes  lactate  dehydrogenase  (LDH)  and  pyru- 
vate  kinase  (PK),  relative  to  values  previously  reported  for  shallow-living  fishes. 
Malate  dehydrogenase  activity  also  was  low,  while  citrate  synthase  (CS)  activity 
was  similar  to  levels  found  in  shallow-living  fishes.  Interspecific  differences  among 
the  rattails  were  large,  especially  for  LDH  activity  which  is  a  strong  indicator  of 
a  fish's  capacity  for  vigorous,  burst  swimming.  Coryphaenoides  armatus,  a  large 
rattail  which  is  likely  to  be  the  most  active  swimmer  among  the  species  studied, 
had  the  highest  enzymic  activities  and  protein  content,  and,  for  LDH,  PK,  and  CS, 
exhibited  a  significant  scaling  of  enzymic  activity  with  body  mass.  Scaling  rela- 
tionships were  not  observed  for  any  other  species.  Brain  enzymic  activities  were 
similar  among  all  species.  Muscle  and  brain  enzymic  activities  also  are  reported 
for  species  belonging  to  four  other  deep-sea  teleost  families.  The  low  levels  of 
enzymes  of  energy  metabolism  found  in  skeletal  muscle  of  these  deep-sea  fish 
species,  and  the  interspecific  variation  in  these  activities  are  discussed  in  terms  of 
the  locomotory  capacities  and  feeding  strategies  of  these  fishes.  The  potential  use- 
fulness of  these  types  of  enzyme  data  in  estimating  whole  fish  respiration  rates  is 
considered.  We  predict  that  the  respiratory  rates  of  the  rattail  species  which  have 
extremely  low  enzymic  activity  levels  will  be  much  lower  than  the  respiratory  rates 
previously  measured  for  C.  armatus. 

INTRODUCTION 

The  macrourid  (rattail  or  grenadier)  fishes  comprise  the  dominant  component 
of  the  bathyal  fish  fauna  in  many  areas  of  the  ocean  (e.g.,  Marshall,  1965,  1973; 
Iwamoto,  1970).  There  are  some  300  macrourid  species,  a  number  of  which  may 
have  cosmopolitan  distributions  (Marshall,  1973;  Iwamoto  and  Stein,  1974).  The 
rattails  are  an  important  component  of  deep-sea  food  webs  (Haedrich  and  Hen- 
derson, 1974;  Pearcy  and  Ambler,  1974).  In  the  area  south  of  New  England,  rattail 
fishes  may  account  for  up  to  80  percent  of  the  slope  megafaunal  biomass  (Haedrich 
and  Rowe,  1977;  Haedrich  et  al.,  1980).  Because  of  their  feeding  habits,  the  rattails 

Received  5  April  1982;  accepted  21  May  1982. 

Abbreviations:  CS,  citrate  synthase;  LDH,  L-lactate  dehydrogenase;  MDH,  L-malate  dehydroge- 
nase; PK,  pyruvate  kinase. 

240 


BIOCHEMISTRY  OF  MACROURID  FISHES  241 

are  important  in  terms  of  energy  input  and  energy  dispersal  in  the  deep  sea  (Hae- 
drich  and  Henderson,  1974;  Pearcy  and  Ambler,  1974;  McClellan,  1976),  and  may 
play  an  important  role  in  the  maintenance  of  macrofaunal  species  diversity  (Dayton 
and  Hessler,  1972;  see  Grassle  and  Sanders,  1973,  for  a  contrasting  view). 

Rattail  species  differ  in  their  feeding  strategies  and,  even  within  a  species,  small 
and  large  individuals  may  differ  in  their  prey  and  location  in  the  water  column. 
Some  rattail  species  are  motile  scavengers,  and  have  been  observed  to  come  to  bait 
(Isaacs,  1969;  Isaacs  and  Schwartzlose,  1975).  Analyses  of  gut  contents  (Haedrich 
and  Henderson,  1974;  Pearcy  and  Ambler,  1974;  McClellan,  1976)  and  head  mor- 
phology (McClellan,  1976)  have  provided  insight  into  their  feeding  habits  and 
sources  of  food.  Smaller  rattail  species,  e.g.,  Nezumia  bairdii,  Coryphaenoides 
(=Lionurus)  carapinus,  and  smaller  individuals  of  other  species,  e.g.,  C. 
(=Nematonurus)  armatus  and  C.  rupestris,  feed  primarily  on  benthic  or  bottom- 
associated  invertebrates  (Haedrich  and  Henderson,  1974;  Pearcy  and  Ambler, 
1974;  McClellan,  1976).  Larger  species,  such  as  C.  rupestris,  C.  armatus,  and  C. 
(  =  Chalinura)  leptolepis  may  rely  more  on  pelagic  organisms,  at  least  once  these 
species  reach  a  certain  size  (Haedrich  and  Henderson,  1974;  Pearcy  and  Ambler, 
1974;  McClellan,  1976).  It  is  not  clear  whether  such  pelagic  prey  are  encountered 
near  the  bottom  or  much  higher  in  the  water  column;  nonetheless,  it  is  likely  that 
these  rattails  make  excursions  into  midwater. 

Rattail  fishes  have  received  relatively  little  physiological  study,  and  we  presently 
have  few  data  concerning  the  physiological  correlates  of  feeding  and  locomotory 
patterns.  Smith  and  Hessler  (1974)  and  Smith  (1978)  have  determined  the  res- 
piration rates  of  two  large,  common  rattail  species  in  situ.  Coryphaenoides  acrolepis 
was  studied  in  the  San  Diego  Trough  at  1230m;  C.  armatus  was  studied  at 
3650  m  in  the  northwest  Atlantic.  Both  species  had  very  low  respiration  rates, 
consuming  oxygen  at  only  approximately  4  percent  of  the  rates  shown  by  similar- 
sized,  shallow-living  related  species  at  the  same  experimental  temperature.  Both 
species  fell  on  a  similar  weight  versus  respiration  rate  curve. 

The  present  experiments  were  initiated  to  obtain  additional  information  about 
the  metabolic  characteristics  of  rattail  fishes,  including  data  on  interspecific  dif- 
ferences in  muscle  metabolism  that  relate  to  variations  in  feeding  strategy  and 
locomotory  capacity.  Our  approach  involved  measurement  of  the  activities  of  key 
enzymes  of  energy  metabolism  (glycolysis  and  the  citric  acid  cycle)  in  white  skeletal 
muscle.  Recent  studies  (Childress  and  Somero,  1979;  Sullivan  and  Somero,  1980; 
Siebenaller  and  Somero,  1982;  Somero,  1982)  have  demonstrated  that  the  levels 
of  activity  of  these  enzymes  in  white  muscle  correlate  strongly  with  the  feeding 
strategy  and  capacity  for  vigorous  swimming  in  a  wide  spectrum  of  marine  fishes. 
Active  pelagic  swimmers  like  tunas  have  up  to  1000-fold  higher  levels  of  glycolytic 
enzyme  activity  per  gram  wet  weight  of  muscle  than  sluggish  deep-sea  species 
(Sullivan  and  Somero,  1980).  Such  enzymic  indices  are  useful  even  in  fine-scale 
comparisons  of  congeneric  fishes  which  differ  in  their  depth  distributions  (Sieben- 
aller and  Somero,  1982).  Thus,  a  shallow-living  scorpaenid  fish,  Sebastolobus 
alascanus,  had  approximately  twice  as  much  activity  for  several  enzymes  of  energy 
metabolism  in  muscle  as  did  a  deep-living,  closely  related  species,  S.  altivelis. 
Interspecific  differences  in  muscle  enzymic  activity  also  correlate  well  with  mea- 
sured variations  in  oxygen  consumption  rate  among  midwater  species  (Childress 
and  Somero,  1979),  a  finding  which  suggests  that  muscle  enzymic  activity  data 
may  be  useful  in  making  predictions  about  in  vivo  metabolic  rates.  Lastly,  glycolytic 
enzymes  of  white  skeletal  muscle  exhibit  a  striking  scaling  relationship  with  body 
size  (Somero  and  Childress,  1980).  Larger  individuals  of  a  species  contain  much 


242  SIEBENALLER,  SOMERO,  AND  HAEDRICH 

higher  levels  of  glycolytic  enzymes  per  gram  muscle  than  smaller  individuals,  a 
scaling  function  which  appears  to  relate  to  the  conservation  of  a  stable  capacity 
for  burst  locomotory  performance  in  all  sizes  of  individuals  of  a  species  (Somero 
and  Childress,  1980).  The  presence  of  this  type  of  metabolic  scaling  relationship, 
therefore,  may  provide  some  clue  as  to  the  importance  of  vigorous  swimming  activity 
in  a  species,  and  may  indicate  whether  large  and  small  members  of  a  species  have 
similar  demands  for  intense  locomotory  performance. 

Our  comparisons  of  different-sized  individuals  of  five  macrourid  species  col- 
lected in  the  northwest  Atlantic  show  that  extremely  large  differences  in  muscle 
enzymic  activity  exist  among  species,  and  among  different-sized  individuals  of  the 
larger,  more  actively  swimming  species.  However,  there  are  no  apparent  differences 
in  muscle  enzymic  activity  among  these  species  related  to  depth  of  occurrence  per 
se.  These  data,  plus  observations  made  on  several  other  deep-living  fishes  collected 
in  the  same  trawls,  are  discussed  in  terms  of  interspecific  differences  in  feeding 
behavior  and  metabolic  requirements  of  life  in  the  deep  sea. 

MATERIALS  AND  METHODS 
Specimens 

Samples  were  taken  with  a  41 -foot  (12.5  m)  Gulf  of  Mexico  shrimp  trawl, 
fished  as  in  Haedrich  et  al.  (1980),  on  cruise  93  of  the  R/V  Oceanus  in  an  area 
south  of  New  England.  Based  on  the  distributional  information  described  in  Hae- 
drich et  al.  (1980),  samples  were  taken  at  appropriate  depth  intervals  to  obtain, 
at  their  depths  of  maximal  abundance,  the  species  used  in  this  study.  Sampling 
was  conducted  in  late  March  and  early  April  so  that  surface  waters  would  be  cold, 
and  specimens  would  not  be  subjected  to  thermal  shock.  The  fishes  often  had  a 
heartbeat  when  brought  to  the  surface,  and  were  maintained  in  ice-cold  seawater 
until  frozen  in  a  — 80°C  freezer  at  sea.  Specimens  were  typically  processed  within 
an  hour  after  the  trawl  was  brought  on  deck.  The  samples  were  transported  to  the 
laboratory  where  they  were  maintained  at  — 76°C. 

A  series  of  five  macrourid  species  encompassing  a  depth  range  of  5000  m  were 
obtained:  Nezumia  bairdii,  Coryphaenoides  rupestris,  Coryphaenoides  (=Lionu- 
rus}  carapinus,  Coryphaenoides  (=Nematonurus}  armatus,  and  Coryphaenoides 
(  =  Chalinura)  leptolepis.  The  depth  ranges  and  depths  of  maximal  abundance  of 
these  species  are  reported  in  Table  I.  Specimens  of  the  following  deep-living  species 
were  also  obtained  and  studied:  Halosauropsis  macrochir  (Halosauridae),  Bath- 
ysaurus  agassizi  (Bathysauridae),  Histiobranchus  bathybius  (Synaphobranchi- 
dae),  and  Dicrolene  intranegra  (Brotulidae).  The  distributions  of  these  species  are 
given  in  Table  III. 

Enzymic  activity  determinations 

The  fish  were  measured  and  weighed.  Tissue  samples  were  dissected  from  the 
frozen  specimens  and  weighed,  and  the  frozen  tissue  was  added  to  an  appropriate 
volume  of  10  mM  Tris-HCl  buffer  (pH  7.5  at  10°C).  For  white  skeletal  muscle, 
the  dilution  was  either  4:1  (volume:weight)  or  8:1,  depending  on  the  viscosity  of 
the  homogenate.  For  brain,  the  dilution  was  8:1.  Tissues  were  homogenized  on  ice 
in  a  ground  glass  tissue  homogenizer  (Kontes  Glass  Co.,  Duall-23  model).  The 
homogenate  was  centrifuged  at  2500  X  g  for  10  minutes  at  4°C.  The  supernatant 
was  used  without  further  purification  for  enzymic  activity  measurements.  All  ac- 
tivities are  expressed  as  ^moles  substrate  converted  to  product  per  minute  per  gram 
wet  weight  of  tissue  at  10°C. 


BIOCHEMISTRY  OF  MACROURID  FISHES 


243 


TABLE  I 

White  skeletal  muscle  compositions  and  enzymic  activity  profiles  of  five  macrourid  fish  species. 


Depth  of 

Mass 

Enzyme  activity  (units/g  wet 

Depth 

maximal 

[mean  & 

wt)  [Mean  ±  S.D.] 

range* 

abundance 

range]             % 

Protein 

N 

(m) 

(m) 

(g)           Water 

(mg/g) 

LDH       PK 

MDH 

CS 

Nezumia  bairdii      8       260-1965 


600 


Corvphaenoides 

rupestris  5       550-1960          1000 

Coryphaenoides 

carapinus  11      1250-2740          2000 

Coryphaenoides 

armatus  13      1885-4815          2900 


Coryphaenoides 

leptolepis  1     2288-4639          3500 


54  81.2 

24-102  ±  1.2  (8) 

84  84.6 

84-86  ±  0.6  (4) 

80  85.3 

23-132  ±0.8(4) 

344  83.7 

34-819  ±  2.4  (9) 

456  82.3 

90-960  ±  0.5  (7) 


144.0  6.9  4.6  17.5  0.62 
±16.5(4)  ±2.7  ±2.2  ±10.1  ±0.15 

142.1  16.0  5.4  9.7  0.58 
±  31.0  (3)  ±  5.8  ±  2.6  ±  0.5  ±  0.10 

119.8  4.7  5.9  6.8  0.50 

±22.5(4)  ±2.4  ±2.2  ±0.9  ±0.19 

177.1  53.1  7.2  18.5  0.79 

±18.2(4)  ±28.9  ±2.4  ±3.5  ±0.26 


144.2 
±  16.5  (4) 


4.3        2.6 
±  1.2    ±  0.3 


6.9          0.41 
±  1.0      ±  0.14 


*  The  depth  ranges  are  from  Haedrich  el  al.,  1980  and  Haedrich,  unpublished  data. 

The  following  enzymes  were  assayed  in  white  skeletal  muscle:  L-lactate  de- 
hydrogenase  (LDH,  EC  1 . 1 . 1 .27;  L-lactate:NAD+  oxidoreductase),  pyruvate  kinase 
(PK,  EC  1.7.1.40;  ATP:  pyruvate  phosphotransferase),  L-malate  dehydrogenase 
(MDH,  EC  1.1.1.37;  L-malate:  NAD+  oxidoreductase),  and  citrate  synthase  (CS, 
EC  4.1.3.7;  citrate:  oxaloacetate  lyase  (CoA-acetylating)).  In  brain  tissue,  LDH, 
PK,  MDH  and  CS  were  assayed  for  some  species.  Assays  were  conducted  as  de- 
scribed in  Somero  and  Childress  (1980).  For  MDH  appropriate  controls  were  run 
to  check  for  the  decomposition  of  oxaloacetate  during  the  course  of  the  experiment. 

Water  and  protein  content  of  white  muscle 

Wet  weights  were  determined  on  muscle  samples,  and  the  samples  were  then 
dried  at  60°C  and  weighed  after  24  hours,  when  they  had  dried  to  a  constant 
weight.  The  percentage  water  was  determined  from  the  difference  between  the 
initial  wet  weight  and  the  final  dry  weight.  Protein  concentration  of  white  muscle 
was  determined  using  the  microbiuret  method  of  Itzhati  and  Gill  (1964).  Homog- 
enates  were  prepared  in  distilled  water  and  diluted  to  100:1  (volume:weight)  with 
NaOH  to  give  a  final  NaOH  concentration  of  1  M.  Samples  were  used  without 
centrifugation.  Protein  concentration  was  determined,  after  addition  of  the  biuret 
reagent,  from  the  difference  in  absorbance  at  310  and  390  nm,  using  bovine  serum 
albumin  as  a  standard. 

RESULTS 
Macrourid  white  skeletal  muscle 

The  enzymic  activities,  and  water  and  protein  contents  of  the  white  skeletal 
muscle  of  the  five  macrourid  species  are  given  in  Table  I.  As  a  group,  these  species 
display  lower  enzymic  activity,  lower  protein  content,  and  higher  water  content 


244 


SIEBENALLER,  SOMERO,  AND  HAEDRICH 


than  do  the  shallower-living  species  which  have  been  studied  (cf.  Childress  and 
Somero,  1979;  Sullivan  and  Somero,  1980).  Lowered  skeletal  muscle  enzymic  ac- 
tivities have  been  observed  for  both  midwater  and  benthopelagic  fishes. 

Within  this  group  of  rattails  there  is  a  wide  variation  of  enzymic  activity  and 
protein  content.  This  among-species  variation  is  not  correlated  with  depth  of  oc- 
currence of  the  species.  Coryphaenoides  armatus  displays  strikingly  higher  levels 
of  protein  and  enzymic  activity  per  gram  wet  weight  of  muscle  than  do  the  other 
species.  Also,  for  C.  armatus,  there  is  a  statistically  significant  scaling  of  enzymic 
activity  to  body  mass  for  CS,  PK,  and  LDH  (Fig.  1).  The  equations  for  these 
scaling  relationships  are:  A  =  1.0  w059*0005  for  CS;  A  =  1.83  W024±013  for  PK, 
and  A  =  1.16  w°66±02°  for  LDH.  The  95%  confidence  intervals  are  given  for  the 


_ 

o 

9 

en 


CD 

>^ 

N 


1.2 
1.0 


4  r 


CS 


• 


j \ i i 


PK 


j i i 


100  - 


50 


LDH 


0   100  200  300  400  500  600  700  800  900 

Body  Mass  (g) 

FIGURE  1 .  The  scaling  of  enzymic  activity  in  white  skeletal  muscle  versus  body  mass  for  individuals 
of  Coryphaenoides  armatus.  Citrate  synthase  (CS),  pyruvate  kinase  (PK)  and  lactate  dehydrogenase 
(LDH)  displayed  statistically  significant  scaling  of  activity  versus  body  mass.  The  equations  fitting  these 
data  are  given  in  Results;  the  lines  shown  were  fit  by  these  equations. 


BIOCHEMISTRY  OF  MACROURID  FISHES  245 


TABLE  II 

Enzymic  activity  in  brain  tissue  of  three  species  of  Coryphaenoides. 


Mass 
[mean  & 

Enzyme  activity  (units/g 
wet  wt)  [Mean±  S.D.] 

range] 

(g) 

LDH 

PK 

MDH 

CS 

Coryphaenoides  rupestris 

66 

27.8 

22.0 

43.5 

2.0 

58-86 

±  4.3 

±  6.7 

±  9.1 

±  0.2 

Coryphaenoides  armatus 

255 

22.0 

13.3 

50.7 

1.4 

67-494 

±  3.7 

±  1.2 

±  5.5 

±  0.2 

Coryphaenoides  leptolepis 

625 

17.6 

13.8 

35.1 

1.3 

278-960 

±  2.6 

±  1.0 

±  4.2 

±  0.2 

Four  individuals  of  each 

species  were  used. 

scaling  exponents.  "A"  is  the  enzymic  activity  and  "W"  the  wet  weight  of  the 
entire  fish  in  grams.  None  of  the  other  macrourids  showed  detectable  mass-related 
scaling  of  enzymic  activity.  For  example,  C.  leptolepis,  for  which  we  had  individuals 
ranging  in  mass  from  90  to  960  grams,  had  a  range  of  LDH  activity  of  only  2.3 
to  5.3  units  per  gram  wet  weight,  with  no  size-related  variation.  The  scaling  patterns 
observed  for  white  muscle  enzymes  of  C.  armatus  agree  with  those  noted  for  a 
variety  of  shallow-living  fishes  (Somero  and  Childress,  1980)  in  that  the  activities 
of  the  two  glycolytic  enzymes,  LDH  and  PK,  increase  with  rising  body  mass,  while 
the  activity  of  the  citric  acid  cycle  (=aerobically  poised)  enzyme  CS  displays  lower 
activity  per  gram  muscle  in  larger  specimens. 

Macrourid  brain  tissue 

The  activities  of  the  four  enzymes  assayed  in  skeletal  muscle  also  were  measured 
in  brain  tissue  of  C.  rupestris,  C.  leptolepis,  and  C.  armatus  (Table  II).  The  values 
are  somewhat  variable,  but  generally  similar  among  the  three  species.  These  ac- 
tivities are  comparable  to  those  reported  for  other  fishes,  both  shallow-  and  deep- 
living  (Childress  and  Somero,  1979;  Sullivan  and  Somero,  1980;  Siebenaller  and 
Somero,  1982).  We  observed  no  scaling  relationships  for  the  brain  enzymes,  but 
this  result  may  be  due  to  the  small  sample  size  used  in  the  study. 

Muscle  enzymic  activities  and  compositions  of  other  deep-sea  families 

The  enzymic  activities  and  water  and  protein  contents  of  white  skeletal  muscle 
of  representatives  of  four  other  deep-sea  fish  families  are  given  in  Table  III.  The 
enzymic  activities  in  these  species  are  low  and  within  the  range  found  for  the 
macrourid  species. 

There  is  variation  among  these  species,  and  wide  variation  between  individuals 
of  Histiobranchus  bathybius.  The  protein  and  water  contents  of  the  muscle  of  this 
species  were  extremely  variable,  and  some  component  of  the  tissue  may  have  caused 
interference  with  the  protein  measurements.  These  data  are  not  reported  here. 

The  number  of  individuals  and  the  size  range  of  individuals  which  were  taken 
in  our  sampling  program  are  not  adequate  to  permit  us  to  address  the  question  of 
mass-related  scaling  in  these  species. 


246 


SIEBENALLER,  SOMERO,  AND  HAEDRICH 


TABLE  III 

White  skeletal  muscle  compositions  and  enzymic  activity  profiles  for  species  of  four  deep-living 
fish  families. 


Depth 

Depth  of 
maximal              Mass 

Enzyme  activity  (units/g 
wet  wt)  [Mean  ±  S.D.] 

range* 

abundance         [mean  & 

% 

Protein 

N               (m) 

(m)              range]  (g) 

Water 

(mg/g) 

LDH 

MDH 

PK 

CS 

Halosauropsis           3          1500-5179 

2300                  290 

81.2 

90.7 

11.6 

3.6 

2.2 

0.40 

macrochir 

248-365 

±  0.4 

±  24.4 

±  1.7 

±0.03 

±  0.4 

±  0.75 

Bathysaurus             2          1500-2967 

2000                  625 

80.9 

107.8 

35.4 

9.2 

11.0 

0.81 

agassizi 

433-817 

±  0.4 

±  11.0 

±  6.0 

±  1.8 

±  0.9 

±  0.21 

Hisliobranchus         2          1885-1093 

2900                  793 

— 

— 

53.0 

8.5 

12.3 

0.61 

bathybius 

328-1258 

±  59.8 

±  7.8 

±  12.6 

±  0.52 

Dichrolene 

intranegra               1             720-1960 

1000                   85 

81.1 

100.5 

46.4 

13.2 

6.4 

1.21 

*  Depth  ranges  are  from  Haedrich  el  at.,  1980,  and  Haedrich,  unpublished  data 


DISCUSSION 

All  of  the  species  examined  in  this  study  have  extremely  low  levels  of  LDH, 
PK,  and  MDH  activity  per  gram  of  skeletal  muscle  compared  to  shallow-living 
fishes.  For  example,  activities  of  LDH,  the  enzyme  which  appears  to  be  the  best 
index  of  a  fish's  capacity  for  intense,  burst  swimming  (Somero  and  Childress,  1 980), 
range  between  approximately  200  and  1000  units  per  gram  in  muscle  of  shallow- 
living,  pelagic  fishes;  and  4  to  150  units  per  gram  in  deep-living  fishes  (Sullivan 
and  Somero,  1980;  Tables  I  and  III).  Citrate  synthase,  an  indicator  enzyme  of  the 
citric  acid  cycle,  is  present  in  only  low  activities  in  white  muscle,  a  reflection  of 
the  anaerobic  poise  of  this  tissue  (cf.  Somero  and  Childress,  1980).  CS  activity 
varies  only  slightly  among  species,  and  only  a  small  reduction  in  CS  activity  is 
noted  in  deeper-living  fishes  (Sullivan  and  Somero,  1980).  MDH  activity  is  inter- 
mediate between  the  two  glycolytic  enzymes  (LDH  and  PK)  and  CS  in  terms  of 
interspecific  variation.  MDH  may  play  some  role  in  cytoplasmic  redox  balance, 
albeit  LDH  is  the  dominant  factor  in  this  context,  and  it  may  also  contribute  to 
the  shuttling  of  reducing  equivalents  between  the  cytosol  and  the  mitochondria, 
and  to  the  function  of  the  citric  acid  cycle.  Because  of  this  variety  of  roles,  MDH 
is  less  apt  to  be  a  strong  indicator  of  burst  swimming  capacity  than  either  LDH 
or  PK.  The  results  of  the  present  study,  like  those  of  earlier  comparisons  of  enzymic 
activities  (Childress  and  Somero,  1979;  Sullivan  and  Somero,  1980;  and  Siebenaller 
and  Somero,  1982),  indicate  that  reduction  in  the  capacity  for  anaerobic  glycolysis 
in  muscle,  i.e.,  in  burst  swimming  ability,  is  a  major  feature  of  adaptation  to  life 
in  the  deep  sea. 

There  is  wide  variation  of  glycolytic  activity  in  white  muscle  among  the  five 
rattail  species,  however,  especially  in  the  case  of  LDH.  The  highest  levels  of  enzymic 
activity,  and  the  only  significant  scaling  relationships  between  enzymic  activity  and 
body  mass,  are  found  for  C.  armatus  (Table  I;  Figure  1).  At  least  larger-sized 
individuals  of  this  species  appear  to  make  excursions  into  midwater  to  prey  on 
pelagic  organisms  (Haedrich  and  Henderson,  1974;  Pearcy  and  Ambler,  1974). 
The  relatively  high  levels  of  glycolytic  enzymes  in  white  muscle  of  C.  armatus,  and 
the  body-mass-related  scaling  noted  for  LDH  and  PK,  may  be  reflections  of  a 
relatively  high  capacity  for  swimming  compared  to  the  other  rattail  species  we 


BIOCHEMISTRY  OF  MACROURID  FISHES  247 

examined.  Coryphaenoides  armatus  also  had  the  highest  muscle  protein  content 
of  all  the  rattails  studied. 

The  second-highest  levels  of  LDH  were  found  in  C.  rupestris.  This  species  has 
a  poorly  ossified  skeleton  and  weak  musculature  development  (Marshall,  1973), 
but  it  has  been  reported  to  make  excursions  into  the  water  column,  and  to  feed  on 
pelagic  prey  (Haedrich,  1974).  Nezumia  bairdii  and  C.  carapinus  feed  on  benthic 
invertebrates  (Haedrich  and  Henderson,  1974;  Pearcy  and  Ambler,  1974;  Mc- 
Clellan,  1976).  Although  larger  individuals  of  C.  leptolepis  may  take  pelagic  prey 
(Pearcy  and  Ambler,  1974),  this  species  has  a  poorly  developed  swimbladder  and 
probably  stays  near  the  bottom  (Marshall,  1973;  Pearcy  and  Ambler,  1974).  These 
species  have  very  low  levels  of  glycolytic  enzymic  activity  in  their  white  skeletal 
muscle,  which  may  reflect  a  low  potential  for  burst  swimming  correlated  with  this 
foraging  habit. 

The  low  levels  of  enzymic  activity  found  in  C.  leptolepis  relative  to  C.  armatus 
demonstrate  that  body  size  does  not  contribute  significantly  to  the  interspecific 
differences  noted  in  enzymic  activity.  Thus,  large  individuals  of  C.  armatus  had 
approximately  ten  times  as  much  LDH  activity  and  three  times  as  much  PK  activity 
as  similar-sized  individuals  of  C.  leptolepis.  The  finding  that  skeletal  muscle  LDH 
activity  is  low  and  very  similar  in  all  sizes  of  C.  leptolepis  examined  indicates  that 
this  fish  is  unlikely  to  have  much  capacity  for  rapid  burst  locomotory  activity.  Burst 
swimming  capacity  would,  in  fact,  decrease  considerably  with  increasing  body  size 
for  C.  leptolepis,  since  a  scaling  relationship  between  body  mass  and  LDH  activity 
comparable  to  that  found  for  C.  armatus  muscle  is  needed  to  conserve  a  constant 
burst  swimming  capacity  as  body  size  increases  (Somero  and  Childress,  1980). 

Smith  (1978)  measured  respiration  rates  in  C.  armatus  that  were  very  low  in 
comparison  to  those  of  shallow-living  fishes  at  similar  temperatures.  Previously, 
Smith  and  Hessler  (1974)  measured  a  comparably  low  respiration  rate  for  C. 
acrolepis.  The  respiration  rate  of  C.  armatus  scaled  with  body  mass  according  to 
the  equation:  Y  =  0.03  W065,  where  Y  is  the  oxygen  consumption  rate  (ml/h)  and 
W  is  the  wet  weight  of  the  fish  (g).  The  scaling  we  have  determined  for  LDH 
activity  in  skeletal  muscle  of  C.  armatus  is  fit  by  a  similar  power  function:  A 
:  1.16  W066,  where  A  is  the  LDH  activity  (units  per  g  wet  weight)  and  W  is  fish 
wet  weight  (g).  The  virtually  identical  scaling  exponents  indicate  a  linear  rela- 
tionship between  oxygen  consumption  rate  and  LDH  activity  in  this  species. 

Childress  and  Somero  (1979)  demonstrated  an  interspecific  correlation  between 
LDH  activity  and  oxygen  consumption  rate  for  midwater  fishes.  A  similar  rela- 
tionship of  oxygen  consumption  rate  and  LDH  activity  for  benthopelagic  rattails 
is  suggested  by  the  scaling  relationships  for  oxygen  consumption  and  LDH  activity 
of  C.  armatus,  as  discussed  above,  and  the  finding  of  similar  levels  of  LDH  activity 
in  C.  acrolepis  and  C.  armatus  (Sullivan  and  Somero,  1980;  Table  I).  The  in  situ 
respiration  rates  of  C.  acrolepis  and  C.  armatus  were  also  similar  (Smith  and 
Hessler,  1974;  Smith,  1978).  However,  the  relationship  of  LDH  activity  and  res- 
piration in  macrourids  does  not  fall  on  the  same  curve  as  the  data  for  midwater 
fishes.  Also,  for  the  midwater  fishes  examined  by  Childress  and  Somero  (1979), 
MDH  activity  correlated  with  oxygen  consumption  rates.  MDH  activity  in  C. 
armatus  does  not  scale  as  the  same  fractional  exponent  of  mass  as  does  oxygen 
consumption,  and  thus  may  not  be  a  predictor  of  respiration  rate  in  macrourid 
species. 

Assuming  a  relationship  between  respiration  rate  and  LDH  activity  in  rattails, 
the  very  low  activities  of  LDH  observed  in  N.  bairdii,  C.  rupestris,  C.  carapinus, 
and  C.  leptolepis  are  indicative  of  extremely  low  rates  of  oxygen  consumption. 


248  SIEBENALLER,  SOMERO,  AND  HAEDRICH 

These  four  rattail  species  may  have  some  of  the  lowest  metabolic  rates  of  any 
fishes.  The  body  mass  versus  respiration  rate  relationship  described  by  Smith  (1978) 
for  C.  acrolepis  and  C.  armatus  may,  therefore,  overestimate  the  oxygen  con- 
sumption rates  of  the  other  rattail  species  we  have  studied. 

Despite  the  wide  interspecific  variation  in  the  activities  of  skeletal  muscle  en- 
zymes, relatively  small  interspecific  differences  were  found  in  comparisons  of  brain 
enzymes  (Table  II).  This  finding  agrees  with  previous  reports  of  Childress  and 
Somero  (1979),  Sullivan  and  Somero  (1980)  and  Siebenaller  and  Somero  (1982), 
who  found  no  evidence  for  depth-  or  activity-related  trends  in  brain  enzymic  ac- 
tivity. The  general  similarity  in  brain  enzymic  activities  for  both  glycolytic  and 
citric  acid  cycle  enzymes  among  widely  different  fishes  from  shallow  and  deep-sea 
habitats  suggests  that  the  requirements  of  neural  function  are  similar  among  dif- 
ferent fishes. 

The  muscle  enzymic  activities  of  the  representatives  of  the  four  other  deep-sea 
fish  families  also  are  very  low  relative  to  shallow-living,  actively  swimming  fishes 
(Table  III;  Sullivan  and  Somero,  1980).  These  low  activities  are  again  likely  to  be 
a  reflection  of  a  relatively  low  capacity  for  active  swimming.  Marshall  (1973) 
considers  the  rattails,  halosaurs  and  brotulids  to  be  slow,  intermittent  swimmers. 
The  low  muscle  enzymic  activities  found  in  H.  macrochir  and  D.  intranegra  and 
the  smaller  rattails  are  consistent  with  this  view.  The  low  skeletal  muscle  activities 
of  the  bathysaur,  B.  agassizi,  also  suggest  a  similar  locomotory  capacity. 

A  high  amount  of  variation  between  individuals  was  noted  for  the  synapho- 
branchid  fish,  Histiobranchus  bathybius.  The  wide  variation  in  muscle  enzymic 
activities  could  be  a  reflection  of  a  strong  scaling  relationship  like  that  noted  for 
C.  armatus;  however,  we  captured  too  few  specimens  of  H.  bathybius  to  test  this 
hypothesis.  The  finding  that  LDH  activity  in  muscle  of  H.  bathybius  reached  95 
units  per  gram  in  the  larger  specimen  examined  (mass  =  1258  g)  and  156  units 
per  gram  in  a  specimen  studied  by  Sullivan  and  Somero  (1980)  (mass  not  known 
due  to  lack  of  a  complete  specimen),  suggests  that  this  species  is  capable  of  an 
active  locomotory  style. 

In  summary,  our  examination  of  five  rattail  fishes  has  revealed  that  depth  of 
occurrence  per  se  is  not  a  factor  in  the  differences  in  white  muscle  enzymic  activities 
within  this  family  of  fishes.  Rather,  the  differences  in  muscle  enzymic  activities 
appear  to  reflect  interspecific  variation  in  feeding  habits.  The  large  rattail,  C. 
armatus,  possessed  the  highest  levels  of  glycolytic  enzymes  and  the  only  scaling 
of  these  activities  with  body  mass.  Both  traits  are  argued  to  be  evidence  for  an 
active  locomotory  habit,  at  least  relative  to  other  rattail  fishes.  The  extremely  low 
muscle  enzymic  activities  found  in  the  species  we  examined  are  taken  as  evidence 
for  very  low  whole  organism  respiration  rates  of  these  fishes.  To  the  extent  that 
whole  organism  oxygen  consumption  rate  is  linearly  related  to  LDH  activity  of 
muscle  (Childress  and  Somero,  1979),  we  propose  that  the  four  rattail  species  found 
to  contain  the  lowest  LDH  activities  have  extraordinarily  low  respiratory  rates, 
rates  that  are  considerably  lower  than  those  which  would  be  predicted  by  extrap- 
olation using  the  respiration  rate  versus  body  mass  relationship  developed  by  Smith 
(1978)  in  his  studies  of  C.  armatus. 

ACKNOWLEDGMENTS 

These  studies  were  supported  by  National  Science  Foundation  grants  PCM80- 
01949  and  PCM80-23166,  and  NSERC  grant  A7230.  We  gratefully  acknowledge 
the  assistance  of  Dr.  Eugene  Copeland,  Marine  Biological  Laboratory,  in  these 
studies. 


BIOCHEMISTRY  OF  MACROURID  FISHES  249 

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Continued  from  Cover  Two 

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- 


CONTENTS 


ANNUAL  REPORT  OF  THE  MARINE  BIOLOGICAL  LABORATORY 1 

Invited  article: 

EVANS,  DAVID  H.,  J.  B.  CLAIBORNE,  LINDA  FARMER,  CHARLES  MALLERY,  AND 
EDWARD  J.  KRASNY,  JR. 
Fish  gill  ionic  transport:  methods  and  models   108 

COPELAND,  D.  EUGENE 

The  anatomy  and  fine  structure  of  the  eye  in  fish.  VI  ciliary  type  tissue 

in  nine  species  of  teleosts  ^. . . . : . . .  YVtTv . .  .\  . . ;  . . . " 131 

DEVINE,  DANA  V.,  AND  JELLE  ATEMA 

Function  of  chemoreceptor  organs  in  spatial  orientation  of  the  lobster, 

Ho  mar  us  americanus'.  differences  and  overlap 144 

FINGER,  THOMAS  E. 

Somatotopy  in  the  representation  of  the  pectoral  fin  and  free  fin  rays 

in  the  spinal  cord  of  the  sea  robin,  Prionotus  carol inus    154 

GLEESON,  RICHARD  A. 

Morphological  and  behavioral  identification  of  the  sensory  structures 
mediating  pheromone  reception  in  the  blue  crab,  Callinectes  sap  id  us    162 

JEBRAM,  DIETHARDT,  AND  BETTY  EVERITT 

New  Victorellids  (Bryozoa,  Ctenostomata)  from  North  America:  the  use 

of  parallel  cultures  in  Bryozoan  taxonomy    172 

LAWN,  I.  D.,  AND  D.  M.  Ross 

The  release  of  the  pedal  disk  in  an  undescribed  species  of  Tealia  (An- 
thozoa:  Actiniaria)    .  .),.Ai:  /.?... .  1 .  A 188 

MALLATT,  JON 

Pumping  rates  and  particle  retention  efficiencies  of  the  larval  lamprey, 

an  unusual  suspension  feeder   ^. , . .  X.  }).  j.  '>.  .,\ 197 

POHLE,  GERHARD,  AND  MALCOLM  TELFORD 

Post-larval  growth  of  Dissodactylus  primitivus  Bouvier,  1917  (Brachy- 
ura:  Pinnotheridae)  under  laboratory  conditions    211 

REED-MILLER,  CHARLENE,  AND  MICHAEL  J.  GREENBERG 

The  ciliary  junctions  of  scallop  gills:  the  effects  of  cytochalasins  and 
concanavalin  A    ~f. -. .  J.'r r. ...  . 225 

SlEBENALLER,  JOSEPH  F.,  GEORGE  N.  SOMERO,  AND  RICHARD  L.  H AEDRK  H 

Biochemical  characteristics  of  macrourid  fishes  differing  in  their  depths 

of  distribution  240 


Volume  163  Number  2 


THE 


BIOLOGICAL  BULLETIN 

- 


- 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 

Editorial  Board 

DANIEL  L.  ALKON,  National  Institutes  of  Health  and      MICHAEL  G.  O'RAND,  Laboratories  for  Cell  Biology, 

Marine  Biological  Laboratory  University  of  North  Carolina  at  Chapel  Hill 

ROBERT  B.  BARLOW,  JR.,  Syracuse  University  R*LPH  S.  QUATRANO,  Oregon  State  University  at 

Corvalhs 

STEPHEN  C.  BROWN,  State  University  of  New  York      JOEL  L  RQSENBAUM,  Yale  University 

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DAVID  H.  EVANS,  University  of  Florida  Laboratory 

HARLYN  O.  HALVORSON,  Brandeis  University  JOHN  D.  STRANDBERG,  Johns  Hopkins  University 

J.  RICHARD  WHITTAKER,  Boston  University 

SAMUEL  S.  KOIDE,  The  Population  Council,  Marine  p         m  and  Marine  Biological  Laboratory 

Rockefeller  University 

E.  O.  WILSON,  Harvard  University 

FRANK  J.  LONGO,  University  of  Iowa  „ 

GEORGE  M.  WOODWELL,  Ecosystems  Center,  Marine 

GEORGE  O.  MACKIE,  University  of  Victoria  Biological  Laboratory 

Editor:  CHARLES  B.  METZ,  University  of  Miami 


OCTOBER,  1982 


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PRINCE  &  LEMON  STS. 
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Editor:  CHARLES  B.  METZ,  University  of  Miami 


OCTOBER,  1982 


Printed  and  Issued  by 
LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 
LANCASTER,  PA. 


The  BIOLOGICAL  BULLETIN  is  issued  six  times  a  year  at  the 
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sylvania. 

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Reference:  Biol.  Bull.  163:  251-263.  (October,  1982) 


ADAPTIVE  SIGNIFICANCE  OF  SEMILUNAR  CYCLES  OF  LARVAL 
RELEASE  IN  FIDDLER  CRABS  (GENUS  UCA):  TEST  OF  AN  HYPOTHESIS 

• 

JOHN  H.  CHRISTY 

Belle  W.  Baruch  Institute  for  Marine  Biology  and  Coastal  Research,  University  of  South  Carolina, 

Columbia.  SC  29208 

ABSTRACT 

The  hypothesis  that  semilunar  timing  of  larval  release  by  fiddler  crabs  (genus 
Uca)  results  in  transport  of  the  final  larval  stage  (megalopa)  by  spring  tide  currents 
to  substrates  in  the  upper  estuary  occupied  by  adults  was  tested  and  rejected.  Water 
temperatures  in  the  North  Inlet  estuary,  South  Carolina,  increased  from  approxi- 
mately 20°C  to  28°C  and  the  length  of  larval  life  decreased  during  the  May-Sep- 
tember breeding  season.  Nevertheless,  ovigerous  female  U.  pugilator,  U.  pugnax, 
and  U.  minax,  collected  bimonthly  and  maintained  in  the  laboratory,  released  larvae 
±  1 .5  d  of  the  full  and  new  moons  throughout  most  of  the  breeding  season.  Megalopae 
of  Uca  spp.  were  most  abundant  in  a  small  tidal  creek  in  the  upper  estuary  during 
nocturnal  flood  tides  and  near  the  bottom  about  5  d  before  and  after  the  spring  tides 
in  September.  Uca  spp.  and  several  other  estuarine  crabs  appear  to  release  larvae 
near  the  times  of  the  high  tides  that  are  followed  by  the  nocturnal  ebb  tides  of 
greatest  amplitude  during  the  semilunar  cycle.  At  North  Inlet,  such  timing  results 
in  rapid  seaward  transport  of  newly  hatched  zoeae  and  subsequent  export  into  coastal 
waters.  Convergence  among  estuarine  brachyurans  in  the  timing  of  larval  release 
probably  reflects  a  shared  adaptive  response  to  selective  factors,  such  as  lethal  com- 
binations of  high  temperatures  and  low  salinities,  or  predation  by  diurnal  plankti- 
vors,  that  cause  high  larval  mortality  during  the  day  in  the  upper  estuary. 

INTRODUCTION 

Semilunar  cycles  of  larval  release  have  been  reported  for  several  estuarine  crabs 
(Christy  and  Stancyk,  1982).  Such  cycles  can  be  inferred  for  17  species  of  fiddler 
crabs  (genus  Uca)  from  cycles  of  male  courtship  activity  (Crane,  1958;  von  Hagen, 
1970;  Christy,  1978;  Zucker,  1978)  female  sexual  receptivity,  mate  choice,  and 
incubation  behavior  (Christy,  1978),  ovarian  and  egg  development  (von  Hagen, 
1962,  1970;  Feest,  1969;  Zucker,  1973;  DeCoursey,  1981)  and  variation  in  the 
density  of  newly  hatched  zoeae  in  the  plankton  (Christy  and  Stancyk,  1982).  Direct 
evidence  of  a  semilunar  hatching  rhythm  has  been  obtained  for  U.  pugnax  under 
laboratory  conditions  (Wheeler,  1978)  and  for  U.  pugilator  in  both  the  laboratory 
and  the  field  (DeCoursey,  1981).  Tidal  and  diel  timing  of  larval  release  has  been 
well  described  for  Uca  pugilator  (DeCoursey,  1979,  1981;  Bergin,  1981),  U.  pugnax, 
and  U.  minax  (DeCoursey,  1979);  all  three  species  release  larvae  at  night  near  the 
time  of  high  tide. 

Hypotheses  concerning  the  ecological  consequences  and  the  adaptive  signifi- 
cance of  semilunar  cycles  of  larval  release  in  Uca  spp.  and  other  estuarine  crabs  fall 
into  two  classes:  those  that  invoke  semilunar  variation  in  factors  such  as  food  avail- 
Received  6  February  1982;  accepted  16  July  1982. 
Contribution  Number  458  of  the  Belle  W.  Baruch  Institute  for  Marine  Biology  and  Coastal  Research. 

251 


252  JOHN  H.  CHRISTY 

ability,  susceptibility  to  predation,  and  tidal  exposure  that  may  effect  the  reproduc- 
tive success  of  adult  males  and  females  (Bergin,  1978,  1981;  Zucker,  1978;  De- 
Coursey,  1979)  and  those  that  rely  on  semilunar  variation  in  factors  that  effect  the 
dispersal,  survival,  and  settlement  rates  of  larvae  (von  Hagen,  1970;  Bergin,  1978; 
Christy,  1978;  Wheeler,  1978;  Zucker,  1978;  DeCoursey,  1979;  Saigusa,  1981).  Al- 
though supporting  evidence  for  some  of  these  hypotheses  has  been  sought  in  inter- 
specific comparisons  of  adult  (Zucker,  1978)  and  larval  (Saigusa,  1981)  ecology,  no 
hypothesis  has  been  critically  tested. 

This  paper  presents  a  test  of  the  hypothesis  that  the  timing  of  larval  release  by 
female  fiddler  crabs  results  in  transport  of  the  final  larval  stage  (megalopa)  by  spring 
tide  currents  to  substrates  suitable  for  settlement  (Christy,  1978).  This  hypothesis 
is  based  on  the  following  observations  and  argument.  Vertical  migration  within  or 
between  larval  stages  relative  to  tidal  and  residual  currents  may  aid  retention  of  crab 
larvae  in  estuaries  (Sandifer,  1975;  Cronin,  1979).  However,  Uca  megalopae  have 
been  found  tens  of  kilometers  from  habitats  occupied  by  adults  in  stratified  estuaries 
where  larval  vertical  migration  should  be  most  effective  in  reducing  seaward  trans- 
port (Dudley  and  Judy,  1971;  Sandifer,  1973).  To  return  to  adult  habitats,  which 
extend  commonly  to  the  heads  (sensu  Carriker,  1967)  of  estuaries  (Crane,  1975), 
megalopae  often  must  move  many  kilometers  up-estuary  before  they  settle  and  molt 
to  crabs.  They  might  do  this  by  remaining  on  or  near  the  bottom  during  ebb  tides 
where  currents  are  weak,  then  rising  in  the  water  column  into  stronger  currents 
during  flood  tides  (Carriker,  1967;  Christy,  1978).  Given  these  patterns  of  transport 
and  behavior,  megalopae  that  could  settle  during  spring  tides,  when  current  velocities 
are  at  a  maximum,  would  return  to  adult  habitats  faster  than  those  that  were  ready 
to  settle  at  other  times.  If  larval  mortality  is  proportional  to  the  time  spent  in  the 
water  column  (Thorson,  1946,  1950;  Vance,  1973),  more  megalopae  that  were  ready 
to  settle  during  spring  tides  would  reach  adult  habitats  than  those  moving  into  the 
upper  estuary  at  other  times;  selection  might  favor  females  that  release  zoeae  that 
become  megalopae  during  spring  tides.  This  idea  appears  consistent  with  the  timing 
of  larval  release  by  U.  pugilator  on  the  southwest  coast  of  Florida  (Christy,  1978). 

The  hypothesis  requires  that  the  time  between  larval  release  and  a  spring  tide 
must  equal  an  integral  multiple  of  the  length  of  larval  development.  Development 
rates  of  brachyuran  larvae  depend  strongly  on  temperature  (e.g.,  Costlow  et  al., 
1960,  1962,  1966;  Vernberg  and  Vernberg,  1975).  On  the  east  coast  of  the  United 
States  Uca  spp.  begin  breeding  when  water  temperatures  are  cool  and  end  breeding 
when  temperatures  are  considerably  higher  (Crane,  1 943).  If  the  hypothesis  is  correct, 
and  if  larval  development  rates  are  temperature  dependent  in  the  field,  then  there 
should  be  a  change  in  the  phase  relationship  between  the  semilunar  cycles  of  larval 
release  and  the  spring  tides  as  water  temperature  increases  seasonally,  and  megalopae 
should  be  transported  to  adult  habitats  only  during  spring  tides.  Therefore,  the 
hypothesis  was  tested  by  monitoring  when  female  Uca  spp.  release  larvae  throughout 
a  breeding  season  and  by  determining  when  megalopae  colonize  adult  habitats. 

MATERIALS  AND  METHODS 

This  study  was  conducted  at  the  North  Inlet  estuary  and  the  Field  Laboratory 
of  the  Belle  W.  Baruch  Institute  for  Marine  Biology  and  Coastal  Research,  George- 
town County,  South  Carolina  (Fig.  1 ).  This  is  a  high  salinity,  homogeneously  mixed 
estuary  in  which  the  currents  are  dominated  by  the  semidiurnal  partial  tide  (Kjerfve 
and  Proehl,  1979). 


LARVAL  RELEASE  IN  FIDDLER  CRABS 


253 


79° 12' 23.5" 


33°21'  19.5"  N 
79°  9'    3.5"  W 


33°  19' 17. 3" 

FIGURE  1.  Northern  portion  of  the  North  Inlet  estuary.  Solid  circles  indicate  the  sites  where  ovi- 
gerous  female  Uca  were  collected.  The  open  circle  shows  the  site  where  the  plankton  was  sampled  for 
crab  megalopae.  The  star  shows  the  location  of  the  tide  gauge.  The  approximate  boundaries  of  the  flood 
and  ebb  deltas  are  indicated  by  the  dashed  lines.  (After  the  North  Inlet  quadrangle  map,  United  States 
Geological  Survey,  1942.  Inlet  morphology  is  approximate.) 


Larval  release  in  the  laboratory 

Cycles  of  larval  release  were  determined  by  maintaining  sequential  collections 
of  ovigerous  females  under  controlled  conditions  and  counting  daily  the  number 
that  released  larvae.  Ovigerous  female  U.  pugilator  and  U.  pugnax  were  dug  from 
intertidal  substrates  (Fig.  1 )  during  low  tide  at  approximately  2-week  intervals  from 
28  July  to  12  September  1978,  and  from  4  May  to  18  September  1979.  Ovigerous 
female  U.  minax  were  collected  concurrently  with  the  other  species  in  1978  only. 
Approximately  100  females  of  each  species  were  collected  in  each  sample.  Female 
crabs  were  maintained  in  the  dark  in  incubation  tubes  suspended  in  filtered  sea 
water  ( 1  /^m,  34%o  salinity)  in  an  insulated  fiberglass  tank  supplied  with  four  under- 
gravel  filters,  two  500-watt  immersion  heaters  controlled  by  a  thermoregulator,  and 
an  opaque  lid.  The  incubation  tubes  allowed  females  to  rest  in  about  0.5  cm  of 
water  and  retained  larvae  after  hatching  (Fig.  2).  From  28  July  to  22  September 
1978  the  temperature  in  the  tank  was  28°C.  During  1979,  the  tank  temperature  was 
24°C  from  4  to  31  May,  26°C  from  1  June  to  29  July,  and  28°C  from  30  July  to 
2  October.  These  temperatures  corresponded  closely  to  the  substrate  temperatures 
at  the  depths  at  which  females  were  collected. 

Each  morning  the  number  of  females  of  each  species  that  released  larvae  was 
recorded  and  assigned  to  the  date  of  the  previous  night.  Every  female  released  all 
her  larvae  in  a  single  night.  The  criteria  for  scoring  release  were  the  absence  of  eggs 
on  the  pleopods  of  females  and  the  presence  of  zoeae  in  the  incubation  tubes.  The 


254 


JOHN  H.  CHRISTY 


Top 
tube 


Female 


Bottom 
tube 


Nylon  cord 
Rubber  band 

Bottom  of  top  tube 

1  mm  mesh 


.Plastic  collar 

glued  to  bottom  tube 


Bottom  of  bottom  tube 

).153  mm  mesh 


tank. 




FIGURE  2.   Incubation  tube  used  to  house  ovigerous  female  Uca  in  a  temperature-controlled  seawater 


latter  criterion  was  necessary  because  females  occasionally,  though  rarely  (5  out  of 
3,015  females),  ate  their  eggs  before  they  hatched. 

Larval  development  rates 

To  test  the  assumption  that  larval  development  rates  depend  on  temperature, 
U.  pugilator  larvae  were  reared  at  22  and  28 °C.  About  1  h  after  hatching,  zoeae 
obtained  from  females  in  the  incubation  tubes  were  mixed  in  a  glass  bowl,  concen- 
trated in  a  light  beam,  and  transferred  by  pipette  into  filtered  sea  water  (0.45  ^m- 
34%o  salinity)  in  the  compartments  of  plastic  boxes  fitted  with  hinged  lids.  Ten  zoeae 
were  placed  into  100  ml  of  water  in  each  compartment.  Zoeae  were  transferred 
daily  to  fresh  seawater  in  clean  compartments  and  fed  a  surfeit  of  1-  to  3-h  old 
Artemia  salina  nauplii.  All  zoeae  were  reared  under  a  14L:10D  cycle  in  an  envi- 
ronmental chamber.  The  22°C  experiment  was  begun  on  the  night  of  1 1  May  1979 
with  230  zoeae  from  9  females.  The  28 °C  experiment  was  begun  on  2  September 
1978  with  160  zoeae  from  12  females.  Zoeae  from  several  females  were  pooled  so 
that  estimates  of  development  rates  would  include  components  of  variation  due  to 
differences  among  broods.  The  water  temperatures  at  high  tide  in  the  marsh  matched 
the  rearing  temperatures  on  the  dates  the  experiments  were  begun. 

Plankton  samples 

To  determine  when  megalopae  reach  the  upper  estuary,  plankton  samples  were 
collected  during  day  and  night  flood  tides  at  a  single  station  near  the  head  of  a  small 


LARVAL  RELEASE  IN  FIDDLER  CRABS  255 

tidal  creek  (1-1.5  m  deep  at  mid-tide)  (Fig.  1).  All  three  species  of  Uca  occur  abun- 
dantly on  the  creek  banks  and  in  the  marsh  adjacent  to  the  sampling  site.  Samples 
were  taken  by  hand  from  a  foot  bridge  with  a  conical  net  (0.5  by  2  m,  800-)um  mesh 
Nitex  cloth)  fitted  with  a  flowmeter. 

From  31  August  to  7  September  1978,  two  to  four  5-min  samples  were  taken 
at  10-min  intervals  beginning  at  mid-flood  tide  (3  h  after  slack  low  water).  For  the 
first  2.5  min  of  each  sample,  the  net  was  suspended  just  above  the  creek  bottom; 
for  the  remaining  2.5  min,  the  net  was  raised  so  that  it  was  just  under  the  surface 
of  the  water.  From  7  to  1 1  September,  two  separate  5-min  top  and  bottom  samples 
were  taken  at  each  mid-flood  tide.  From  12  to  23  September,  one  top  and  one 
bottom  sample  were  taken.  Finally,  on  20  and  21  September,  one  top  and  one 
bottom  sample  were  taken  during  mid-ebb  tide  (3  h  after  slack  high  water)  at  night. 
From  10  to  25  m3  (mean  ±  SD  =  15.44  ±  4.90  m3)  of  water  was  filtered  during 
each  5-min  sample.  All  Uca  megalopae  were  counted  in  each  sample.  Megalopal 
densities  are  the  means  of  the  densities  of  the  sequential  samples  collected  during 
each  sampling  period. 

Physical  measurements 

From  19  July  to  4  October  1978  and  from  1 1  January  to  23  July  1979  surface 
water  temperatures  were  measured  within  1  h  of  the  time  of  a  high  tide  at  or  near 
the  site  where  the  plankton  samples  were  taken.  From  1 1  January  to  30  April  1979 
water  temperatures  were  recorded  during  the  day.  At  all  other  times  temperatures 
were  taken  at  night.  On  two  occasions  temperatures  were  measured  within  30  min 
of  slack  high  water  at  several  points  along  a  transect  from  the  mouth  of  North  Inlet 
to  the  upper  marsh  and  were  found  to  vary  less  than  1.5°C. 

Tide  heights  and  amplitudes  were  obtained  from  a  tide  gauge  located  in  the 
North  Inlet  estuary  (Fig.  1).  This  paper  relates  semilunar  cycles  of  larval  release  to 
semilunar  cycles  in  the  amplitude  of  nocturnal  ebb  tides.  A  nocturnal  ebb  tide  is 
defined  as  one  that  follows  a  high  tide  that  occurs  between  the  hours  of  sunset  and 
sunrise.  When  both  high  tides  occurred  during  daylight,  the  one  closest  to  sunrise 
or  sunset  was  designated  as  the  nocturnal  high  tide  for  that  day. 

RESULTS 
Breeding  seasonally  and  water  temperatures 

Crab  activity  was  observed  daily  during  low  tide  throughout  the  1979  breeding 
season.  Male  U.  pugilator  first  courted  on  1 3  March.  By  the  end  of  March,  both  U. 
pugilator  and  U.  pugnax  were  courting,  and  both  species  fed  during  low  tide  in 
aggregations  in  the  lower  intertidal  zone.  Courtship  activity  declined  rapidly  after 
the  full  moon  on  16  September  1978  and  after  the  new  moon  on  21  September 
1979,  and  ended  for  the  year  about  5  d  after  both  dates. 

Water  temperature  increased  rapidly  in  the  spring  from  a  low  of  about  7°C  in 
February  to  about  20°C  by  late  April  and  early  May.  Temperatures  continued  to 
rise  to  about  28 °C  in  the  late  summer  (Fig.  3). 

The  length  of  incubation  in  summer  for  several  species  of  Uca  is  from  1 2  to  15 
d  (Feest,  1969;  von  Hagen,  1970;  Greenspan,  1975;  Christy,  1978).  If  females  mated 
and  began  incubation  in  late  March  and  cool  spring  temperatures  no  more  than 
double  the  period  of  incubation,  then  females  may  have  first  released  larvae  in  late 
April.  Larval  release  by  U.  pugilator  first  occurred  during  the  period  of  2  to  6  May 
in  1981  (J.  Christy,  unpublished).  Stage  I  Uca  zoeae  were  common  in  plankton 


256 


JOHN  H.  CHRISTY 


28- 

Ml"'1 

24 

0° 

4  *      '                    i 

^  20- 

3 

"S 

M 

55   16- 

Q. 
E- 

4 

H   12- 

I 

8- 

tM 

J          FMAMJ          J          A          S         0 

Month 

FIGURE  3.  Bimonthly  means  of  daily  water  temperatures  in  the  North  Inlet  estuary  recorded  within 
1  h  of  diurnal  (1  1  January-30  April)  or  nocturnal  (1  May-4  October)  high  tides.  Temperatures  were 
recorded  in  1978  (19  July-4  October)  and  in  1979  ( 1 1  January-23  July).  The  bars  indicate  two  standard 
errors  above  and  below  each  mean. 

samples  taken  at  the  study  site  in  mid-May,  1979  (Christy  and  Stancyk,  1982).  At 
the  beginning  of  the  breeding  season,  Uca  larvae  probably  develop  in  water  averaging 
20  to  22°C. 

Larval  development  rates 

Development  of  U.  pugilator  zoeae  was  significantly  slower  at  22°C  (mean 
±  SD  ==  19.4  ±  2.47  d,  range  ==  16-27  d,  N  =  230,  76%  survival  to  megalopa)  than 
at  28°C  (mean  ±  SD  =  14.7  ±  2.39  d,  range  =  12-20  d,  N  =  160,  59%  survival  to 
megalopa)  (/267  =:  19.013,  P  <g  0.001).  These  experiments  confirm  that  development 
rates  of  Uca  larvae  are  temperature  dependent  in  the  laboratory  (Vernberg  and 
Vernberg,  1975).  On  the  assumption  that  larval  development  rates  also  depend  on 
temperature  in  the  field,  it  seems  justified  to  expect,  if  the  hypothesis  is  correct,  a 
seasonal  change  in  the  timing  of  larval  release  relative  to  the  spring  tides  as  water 
temperatures  increase  during  the  breeding  season. 

Larval  release  in  the  laboratory 

During  August  and  September  1978,  U.  pugilator,  U.  pugnax,  and  U.  minax 
displayed  marked  semilunar  cycles  of  larval  release  (Fig.  4)  as  did  U.  pugilator  and 
U.  pugnax  during  most  of  the  1979  breeding  season  (Fig.  5).  Females  of  all  three 
species  released  larvae  (mean  ±  SD)  0.06  ±  1. 1 16  d  before  the  date  of  a  full  or  new 
moon  (Table  I).  In  1978  the  average  deviations  of  the  mean  dates  of  release  from 
the  dates  of  the  syzygies  were  0.24  d  for  U.  pugilator,  —0.09  d  for  U.  pugnax,  and 
-0.80  d  for  U.  minax.  In  1979  U.  pugilator  released  larvae,  on  average,  0. 19  d  and 
U.  pugnax  0.41  d  after  a  full  or  new  moon.  There  was  no  significant  correlation 
between  the  mean  date  of  larval  release  for  each  cycle  relative  to  the  date  of  the  full 
or  new  moon  and  the  sequential  rank  of  the  date  of  each  syzygy  during  the  breeding 
season  for  either  U.  pugilator  [Kendall's  coefficient  of  rank  correlation  (Sokal  and 
Rohlf,  1969);  r  ==  0.36,  N  =  8,  P  >  0.05]  or  U.  pugnax  (r  =  0.27,  N  =  8,  P  >  0.05). 
The  expected  seasonal  change  in  the  timing  of  larval  release  relative  to  the  full  and 
new  moons  and  spring  tides  did  not  occur. 


LARVAL  RELEASE  IN  FIDDLER  CRABS 


257 


1      5 


10     15     20     25     30 
August 


5      10     15     20 
September 


FIGURE  4.  Cycles  of  larval  release  by  Uca  spp.  in  the  laboratory  in  1 978.  The  triangles  show  the 
dates  on  which  each  sample  of  ovigerous  females  was  collected.  The  dates  of  the  full  and  new  moons 
are  indicated  by  the  open  and  solid  circles,  respectively. 

Variation  in  the  density  of  Uca  megalopae 

Uca  megalopae  were  significantly  more  abundant  in  the  water  column  during 
the  night  than  during  the  day  [Fig.  6;  Wilcoxon's  signed  ranks  test  comparing  the 


10        20         3O         10        20        30          10 


August  September 

O        •        O 


FIGURE  5.  Cycles  of  larval  release  by  Uca  spp.  in  the  laboratory  in  1 979  and  the  amplitudes  of  the 
nocturnal  ebb  tides  at  North  Inlet.  Dots  on  the  x-axis  indicate  that  ovigerous  females  were  present  in  the 
laboratory  but  none  released  larvae.  Moon  phases  are  indicated  as  in  Figure  4. 


258  JOHN  H.  CHRISTY 

TABLE  I 

Deviation  of  the  mean  date  (±SD,  days)  of  larval  release  in  the  laboratory  from  the  date 
of  a  full  or  new  moon. 


Date  of 
syzygy 

U.  pugilator 

U.  pugnax 

U.  mina\ 

Mean  date 

TV 

Mean  date 

N 

Mean  date               N 

1978 

Aug.     3 

1.40  ±  2.29 

99 

-0.26  ±  1.46 

34 

-0.65  ±  1.75            95 

Aug.   18 

-1.42  ±  6.90 

100 

-1.58  ±  1.60 

89 

-1.06  ±  1.46            83 

Sept.    2 

-0.69  ±  2.20 

100 

-1.43  ±  2.10 

100 

-0.69  ±  1.48            35 

1979 

May   11 

-0.38  ±  4.28 

99 

1.82  ±  5.21 

51 

May  25 

-0.30  ±  6.80 

100 

-1.29  ±  4.26 

82 

June  10 

-0.62  ±  2.07 

110 

0.12  ±  2.53 

102 

June  24 

-0.54  ±  3.20 

100 

-0.37  ±  2.06 

116 

Julv     9 

1.41  ±  2.95 

97 

1.24  ±  2.40 

109 

July    23 

2.27  ±  7.08 

115 

0.57  ±  2.03 

114 

Aug.     7 

0.06  ±  1.96 

118 

-0.63  ±  1.61 

115 

Sept.    6 

-0.20  ±  3.98 

104 

1.84  ±  3.14 

96 

density  of  megalopae  in  each  nocturnal  flood  tide  and  subsequent  diurnal  flood  tide 
(Sokal  and  Rohlf,  1969);  T  11,  N ----  17,  P<  0.005]  and  significantly  more  (80% 
on  average)  were  moving  in  the  lower  50  cm  of  the  water  column  at  mid-flood  tide 
than  near  the  surface  (Wilcoxon's  signed  rank  test  comparing  densities  each  night 
in  surface  and  bottom  samples;  T  --=  50,  N  =  38,  P  <  0.005).  On  19  and  20  September 
densities  of  megalopae  were  2.02  and  3.31  per  m3  during  nocturnal  flood  tides.  In 
contrast,  approximately  6  h  later  during  nocturnal  ebb  tides,  densities  had  dropped 
to  0.09  and  0.02  megalopae  per  m3  on  the  two  nights,  respectively.  Megalopae  were 
most  abundant  about  5  d  before  and  after  the  spring  tides  (Fig.  6),  not  during  the 
spring  tides  as  would  be  expected  if  the  hypothesis  were  correct. 

DISCUSSION 

Semilunar  cycles  of  larval  release  by  U.  pugilator  and  U.  pugnax  in  the  laboratory 
in  1979  corresponded  closely  to  semilunar  cycles  in  the  density  of  newly  hatched 
stage  I  zoeae  in  the  upper  estuary  (Christy  and  Stancyk,  1982  This  indicates  that 
the  timing  of  larval  release  in  the  laboratory  probably  accurately  estimates  the  timing 
of  larval  release  in  the  field. 

Neither  U.  pugilator  nor  U.  pugnax  exhibited  a  semilunar  cycle  of  larval  release 
during  May  1979.  Wheeler  (1978)  reported  a  similar  aperiodicity  in  June  for  U. 
pugnax  collected  in  Delaware  and  maintained  in  the  laboratory.  Sesarma  cinereum 
from  North  Inlet  also  lacked  cycles  of  reproduction  and  hatching  in  the  field  and 
laboratory  during  May  and  June  in  both  1978  and  1979,  though  this  crab  exhibited 
marked  semilunar  cycles  of  larval  release  at  other  times  (Dollard,  1980).  The  causes 
and  consequences  of  aperiodic  larval  release  by  these  crabs  in  the  early  breeding 
season  are  unknown. 

The  timing  of  larval  release  by  U.  pugilator  and  U.  pugnax  changed  little  during 
the  1979  breeding  season  even  though  water  temperatures  increased  at  least  5°C. 
The  rearing  experiments  confirmed  that  the  length  of  larval  development  decreases 
with  an  increase  in  temperature.  If  the  larval  lifespan  in  the  field  decreased  as  the 
water  temperature  increased  from  May  to  September,  then  it  is  clear  that  females 


LARVAL  RELEASE  IN  FIDDLER  CRABS 


259 


10.00  - 


<D 

a. 

0) 

<o 
o. 
o 

<a 

O) 

o> 

E 

<a 
o 


1.00- 


0.10- 


0.01 


€ 


O 


246 
September 


8 


10     12     14      16     18     20    22 
Date 


FIGURE  6.  Temporal  variation  in  the  density  of  Uca  spp.  megalopae  during  September  1978. 
Samples  were  collected  during  daytime  (broken  bars)  and  nighttime  (solid  bars)  flood  tides  in  a  tidal  creek 
in  the  upper  estuary  (Fig.  1).  Densities  of  0  megalopae  per  m3  are  indicated  by  dots  on  the  x-axis.  Full 
and  new  moons  are  indicated  as  in  Figure  4.  First  and  last  quarter  moons  are  indicated  by  half-solid 

circles. 


did  not  time  release  so  that  megalopae  would  be  present  and  ready  to  settle  during 
spring  tides  at  all  times  in  the  breeding  season. 

Megalopae  were  expected  to  reach  the  upper  estuary  during  spring  tides,  but 
little  movement  occurred  at  such  times.  The  observed  bimodal  distribution  of  the 
abundance  of  megalopae  during  the  semilunar  cycle  may  reflect  differential  rates 
of  larval  development  or  transport  among  the  three  species  of  Uca,  or  temporal 
variation  in  hydrographic  features  that  affected  the  transport  of  all  species  equally. 
Megalopae  may  have  been  rare  during  the  syzygies  because  the  tide  ebbs  during 
most  of  the  night  around  the  time  of  the  full  and  new  moons  in  the  North  Inlet 
estuary  and  because  megalopae  entered  the  water  column  primarily  during  nocturnal 
flood  tides.  Any  hypothesized  selective  advantage  to  megalopae  that  move  up  the 
estuary  during  spring  tides  must  be  less  than  the  advantages  to  megalopae  that  move 
only  during  the  night. 

During  each  summer  tide  cycle  there  is  a  net  export  of  approximately  1 5%  of 
the  brachyuran  crab  larvae  (99%  stage  I)  that  are  entrained  in  the  water  that  is  tidally 
pumped  across  the  boundaries  of  the  North  Inlet  estuary  (Christy  and  Stancyk, 
1982).  Crab  larvae  that  develop  in  the  ocean  may  enter  the  estuary  by  being  trans- 
ported landward  by  currents  near  the  bottom  (Scheltema,  1975;  Sulkin  et  «/.,  1980). 
This  study  suggests  that  once  Uca  megalopae  occur  in  tidal  creeks,  they  selectively 
ride  flood  tides  at  night,  perhaps  moving  in  a  saltatorial  fashion  to  substrates  in  the 
upper  estuary.  Uca  megalopae  were  common  on  intertidal  substrates  during  diurnal 
low  tides  following  nights  of  peak  abundance,  but  they  were  rare  when  few  were 


260  JOHN  H.  CHRISTY 

caught  the  previous  night.  Megalopae  that  moved  past  the  sampling  site  at  night 
probably  were  seeking  substrates  on  which  to  settle. 

The  results  of  this  study  do  not  support  the  hypothesis  that  cycles  of  larval  release 
by  Uca  spp.  result  in  maximum  rates  of  transport  of  megalopae  by  spring  tide 
currents  to  substrates  in  the  upper  estuary.  Rather,  the  timing  of  larval  release 
appears  to  result  in  rapid  seaward  transport  of  newly  hatched  zoeae  on  nocturnal 
ebb  tides. 

At  North  Inlet,  the  amplitude  of  nocturnal  ebb  tides  is  correlated  with  the  semilu- 
nar  cycle.  High  tides  occur  just  after  sunset  during  the  full  and  new  moons  and  the 
subsequent  ebb  tides  are  greater  in  amplitude  than  those  that  occur  at  other  times 
in  the  semilunar  cycle  (Fig.  5).  Since  U.  pugilator,  U.  pugnax,  and  U.  minax  release 
larvae  only  at  night  near  the  time  of  high  tide,  and  since  peak  hatching  occurred 
near  the  time  of  the  full  and  new  moons,  these  crabs  were  releasing  larvae  at  the 
time  in  the  semilunar  cycle  when  stage  I  zoeae  would  be  transported  most  rapidly 
at  night  toward  the  ocean.  It  is  impossible  to  judge  whether  larval  release  occurs  in 
response  to  factors  that  vary  with  the  phases  of  the  moon  or  in  response  to  factors 
that  vary  with  the  amplitude  of  nocturnal  ebb  tides  because  these  two  cycles  coincide 
at  North  Inlet.  To  distinguish  between  these  alternatives,  one  needs  to  know  when 
larval  release  occurs  at  a  site  where  nocturnal  ebb  tides  of  greatest  amplitude  occur 
sometime  other  than  during  the  syzygies. 

Reproductive  cycles  have  been  described  for  17  species  of  Uca  (von  Hagen, 
1962,  1970;  Feest,  1969;  Zucker,  1973,  1978;  Christy,  1978;  Wheeler,  1978;  De- 
Coursey,  1 98 1 )  at  sites  ranging  from  the  east  coast  of  India  to  the  mid-Atlantic  coast 
of  the  United  States.  With  one  exception,  both  larval  release  and  the  greatest  am- 
plitude nocturnal  ebb  tides  during  the  semilunar  cycle  occur  within  about  three  days 
of  the  full  and  new  moons.  However,  in  Charlotte  Harbor  on  the  west  coast  of 
Florida,  both  larval  release  by  U.  pugilator  and  nocturnal  ebb  tides  of  maximum 
amplitude  usually  occur  during  the  quarter  moons  (Christy,  1978;  NOAA  tide  ta- 
bles). This  exception,  which  provides  the  only  data  of  use  to  distinguish  between 
the  above  alternatives,  suggests  larval  release  is  timed  to  occur  during  large  amplitude 
ebb  tides  at  night,  not  during  a  particular  phase  of  the  moon. 

Semilunar  cycles  of  larval  release  have  been  described  for  5  species  of  grapsid 
crabs  (Warner,  1967;  Saigusa  and  Hadaka,  1978;  Seiple,  1979;  Dollard,  1980),  2 
gecarcinids  (Gifford,  1962;  Henning,  1975;  Klaasen,  1975),  a  xanthid  and  a  pin- 
notherid  (Christy  and  Stancyk,  1982).  All  these  estuarine  crabs  release  larvae  on 
large  amplitude  nocturnal  ebb  tides  near  the  time  of  the  full  and  new  moons.  It  is 
unlikely  that  convergence  in  such  a  fundamental  feature  of  the  reproductive  ecology 
of  these  terrestrial,  semiterrestrial,  and  benthic  crabs  is  a  fortuitous  result  of  similar 
adaptive  responses  to  selective  factors  that  operate  differently  in  the  diverse  habitats 
of  the  adults  (e.g.,  Zucker,  1978;  DeCoursey,  1979;  Bergin,  1981).  Convergence 
among  these  species  more  likely  reflects  a  shared  adaptive  response  to  mortality 
factors  experienced  in  common  by  their  meroplanktonic  larvae  (but  see  Saigusa, 
1981).  This  study  suggests  such  selective  factors  must  cause  higher  larval  mortality 
during  the  day  and  in  the  upper  estuary  than  during  the  night  and  in  the  lower 
estuary  or  offshore.  The  following  mortality  factors  may  meet  these  criteria. 

On  the  Atlantic  and  Gulf  Coasts  of  the  United  States,  larval,  postlarval,  and 

juvenile  stages  of  many  marine  and  estuarine  spawned  fish  use  the  upper  reaches 

of  tidal  creeks  as  "nurseries,"  moving  seaward  as  they  grow  and  mature  (e.g.,  Chao 

and  Musick,  1977;  Bozeman  and  Dean,  1979;  Shenker  and  Dean,  1979;  Weinstein, 

1979).  Planktivorous  species  such  as  menhaden  (Brevoortia  tyrannus),  silversides 


LARVAL  RELEASE  IN  FIDDLER  CRABS  26 1 

(Menidia  menidid)  and  the  bay  anchovie  (Anchoa  mitchilli)  are  abundant  in  the 
upper  estuary  during  the  entire  crab  breeding  season.  Other  common  species  such 
as  spot  (Leiostomus  xanthurus)  and  pinfish  (Lagodon  rhomboides)  are  planktivorous 
only  when  small,  in  the  spring,  when  crabs  begin  to  breed  (Thayer  et  al.,  1974; 
Kjelson  et  al.,  1975;  Chao  and  Musick,  1977).  In  general,  such  species  feed  on 
planktonic  crustaceans  only  during  the  day  (e.g.,  Kjelson  et  al.,  1975;  Robertson 
and  Howard,  1978).  Predation  in  the  upper  estuary  by  diurnally  feeding  planktivores 
may  produce  powerful  selection  on  when  estuarine  crabs  release  larvae.  Zoeae  that 
are  rapidly  transported  seaward  following  release  near  the  peaks  of  large  amplitude 
nocturnal  tides  may  better  escape  such  predation  than  those  released  at  other  times 
(see  also  Bergin,  1978).  If  true,  one  would  expect  crab  larvae  that  complete  devel- 
opment in  the  upper  estuary  to  possess  traits  that  reduce  predation. 

Rhithropanopeus  harrisi  is  found  in  the  extreme  upper-reaches  of  tidal  creeks 
and  along  rivers  at  the  headwaters  of  estuaries  from  New  Brunswick  to  Brazil 
(Williams,  1965).  This  xanthid  crab  releases  larvae  continuously,  its  zoeae  display 
patterns  of  vertical  migration  that  minimize  seaward  transport,  and  it  completes 
larval  development  in  the  estuary  (Williams,  1971;  Cronin,  1979).  R.  harrisi  zoeae 
are  striking  among  the  xanthid  larvae  that  are  common  in  estuaries  on  the  western 
Atlantic  coast  because  they  possess  extremely  long  rostral  and  antennal  spines 
(Chamberlain,  1962;  Kurata,  1970).  Recent  experiments  demonstrate  that  these 
spines,  together  with  the  dorsal  spine,  deter  ingestion  by  small  planktivorous  fish 
because  they  make  zoeae  too  large  (approximately  2  mm)  to  enter  their  buccal 
cavities  (Morgan,  1981). 

Dollard  (1980)  suggested  that  larvae  released  on  nocturnal  high  tides  might 
escape  lethal  high  temperatures.  Maximum  temperatures  are  likely  to  occur  during 
late  afternoon  low  tides  in  shallow  tidal  creeks  in  the  upper  estuary.  At  North  Inlet 
in  July  and  August  such  temperatures  commonly  exceed  40°C  (Dollard,  1980), 
while  maximum  temperatures  in  deeper  channels  in  the  middle  and  upper  estuary 
remain  around  30  to  32°C  (Bergin,  1978). 

Survival  rates  at  high  temperatures  of  first  stage  Uca  sp.  zoeae  depend  on  salinity 
(Vernberg  and  Vernberg,  1975).  Fifty  percent  of  stage  I  U.  pugilator  zoeae  die  within 
1  h,  while  fifty  percent  of  U.  pugnax  zoeae  die  within  5  h  at  40°C  and  20%o.  At 
35%o  and  40°C,  fifty  percent  mortality  of  U.  pugilator  and  U.  pugnax  zoeae  occurs 
at  12.5  and  6.5  h,  respectively.  At  38°C  zoeae  of  both  species  survive  over  2.5  d  at 
20%o  and  about  3.5  d  at  35%o.  Comparable  data  for  the  other  crabs  discussed  above 
are  not  available. 

During  summer  low  tides,  larval  mortality  will  be  highest  in  hot,  low  salinity 
water  in  pools  and  shallow  creeks  in  the  upper  marsh.  Zoeae  that  are  released  at 
high  tide  just  after  dark  would  be  transported  seaward  and  might  experience  rela- 
tively high  salinities  and  cool  morning  temperatures  during  their  first  low  tide.  By 
migrating  vertically  with  respect  to  flood-  and  ebb-directed  currents,  zoeae  might 
be  further  displaced  seaward  into  cool  high-salinity  water  during  subsequent  tidal 
cycles.  Larvae  that  are  released  at  the  peak  of  large  amplitude  tides  that  occur  after 
sunset  may  best  escape  lethal  combinations  of  salinity  and  temperature  in  both 
space  and  time  (see  also  Saigusa,  1981). 

I  emphasize  that  future  hypotheses  about  the  adaptive  significance  of  the  timing 
of  larval  release  in  Uca  spp.  and  many  other  estuarine  brachyurans  must  explain 
why  these  crabs  release  larvae  at  high  tide  at  night  when  the  amplitude  of  the 
nocturnal  ebb  tides  are  at  a  semilunar  maximum  and  zoeae  are  most  rapidly  trans- 
ported away  from  the  upper-reaches  of  the  estuary. 


262  JOHN  H.  CHRISTY 

ACKNOWLEDGMENTS 

I  thank  Drs.  D.  Allen,  P.  Feeny,  M.  Salmon,  and  S.  Stancyk  for  their  comments 
on  a  draft  of  this  paper.  Dr.  Pat  DeCoursey  assisted  with  the  initiation  of  this  research 
and  shared  her  knowledge  and  unpublished  data  on  tidal  and  diel  timing  of  larval 
release  by  Uca  sp.  The  staff  of  the  Baruch  Institute  provided  frequent  and  capable 
assistance.  This  research  was  supported  by  Grant  OCE77-20960  from  The  National 
Science  Foundation. 

» 

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ECHINODERM  CALCITE:  A  MECHANICAL  ANALYSIS 
FROM  LARVAL  SPICULES 

RICHARD  B.  EMLET 

Department  of  Zoology.  University  of  Washington,  Seattle,  WA  98195,  and  Friday  Harbor 
Laboratories,  University  of  Washington,  Friday  Harbor,  WA  98250 

ABSTRACT 

The  flexural  stiffness  (El )  was  measured  for  simple  and  fenestrated  spicules  in 
echinoid  larvae.  A  Young's  modulus  (E)  of  36.3  X  109  N/nr  was  estimated  for 
these  calcitic  spicules  by  El /I  where  /  was  calculated  independently  from  measure- 
ments made  by  SEM.  The  flexural  stiffness  of  fenestrated  spicules  is  approximately 
three  times  greater  than  that  of  simple  spicules.  This  increased  flexural  stiffness  is 
due  to  structural  and  not  material  differences  between  the  spicules.  At  the  material 
level,  this  calcitic  tissue  behaves  like  a  composite  which  will  reduce  stiffness  but 
increase  strength  compared  to  inorganic  calcite.  At  the  structural  level  its  porous 
nature  increases  its  stiffness  and  buckling  strength  over  that  of  a  solid  structure  of 
similar  weight.  These  characteristics  should  also  increase  the  tensile  strength  of  this 
skeletal  component  and  increase  its  usefulness  as  a  strong,  stiff  element  in  most 
echinoderm  skeletons. 

INTRODUCTION 

This  paper  discusses  the  mechanical  properties  of  echinoderm  calcitic  tissue 
(hereafter  echinoderm  calcite)  at  the  material,  structural,  and  skeletal  levels.  I  present 
results  of  bending  studies  on  simple  and  fenestrated  spicules  of  echinoid  larvae. 
These  spicules  have  the  same  composition  and  manner  of  formation  as  adult  calcitic 
structures  (e.g.,  Okazaki  and  Inoue,  1976;  Loeper  and  Pearse,  1981).  The  simple 
shape  of  larval  spicules  facilitates  the  measurement  of  the  mechanical  properties  of 
echinoderm  calcite.  The  results  provide  evidence  that  echinoderm  calcite  is  a  com- 
posite material  and  exemplify  the  consequences  of  arranging  this  material  into  po- 
rous structures.  In  the  discussion  I  generalize  the  findings  of  this  study  to  adult 
structures  and  propose  new  ideas  which  may  help  explain  the  unusual  structure  of 
echinoderm  calcite.  This  treatment  expands  the  known  mechanical  role  of  echi- 
noderm calcite  beyond  withstanding  compressive  loads. 

MATERIALS  AND  METHODS 

Echinoplutei  with  simple  and  fenestrated  skeletons  were  reared  in  culture  or 
obtained  from  the  plankton  near  Friday  Harbor,  Washington.  Culture  methods  are 
adapted  from  Hinegardner  (1967)  and  Strathmann  (1971).  Spicules  from  larvae  of 
Stronglyocentrotus  droebachiensis  O.  F.  Muller,  Strongylocentrotus  franciscanus  A. 
Agassiz,  and  Dendraster  excentricus  Eschscholtz  were  isolated  with  5%  sodium  hy- 
pochlorite  (Chlorox  Bleach)  and  washed  three  times  with  distilled  water.  Adult  cal- 
citic structures  from  the  holothurian,  Psolus  chitonoides  H.  L.  Clark  were  isolated 
by  a  similar  method. 

Received  24  March  1982;  accepted  16  July  1982. 

264 


ECHINODERM  SKELETAL  MECHANICS  265 

Definitions  of  mechanical  terms  are  as  follows: 

a:       Stress  —  force/cross-sectional  area,  where  the  force  acts  over  that  area.  Units: 

N/m2. 
e:        Strain  —  change  in  length/original  length,  where  change  in  length  is 

produced  by  a  stress.  Units:  dimensionless  or  m/m. 
E:      Young's  modulus  —  the  stiffness  of  a  material,  a/€.  The  stress  in  a  material  is 

divided  by  the  strain  produced  under  stress.  Units:  N/m2. 
/:       Second  moment  of  area  of  a  cross-section  is  a  description  of  the  geometric 

distribution  of  material  around  a  neutral  axis  of  bending.  /  =  J  y2  dA  where 

dA  is  area  of  material  at  distance  y  from  the  neutral  axis  (see  Wainwright  et 

al,  1976).  For  a  circular  cross-section  /  =  1/4  vrr4  where  r  =  radius.  Units: 

m4. 
El:     Flexural  stiffness  of  a  structure  —  the  product  of  Young's  modulus  and  the 

second  moment  of  area  which  describes  the  ability  of  a  structure  to  resist 

bending.  Units:  Nm2. 

El  may  be  used  to  compare  structures  which  vary  in  material  or  shape,  and  is 
especially  useful  when  shapes  are  complex.  If  either  E  or  I  and  flexural  stiffness  are 
known,  then  the  other  variable  may  be  determined.  In  this  study  El  is  determined 
for  spicules  from  small  scale  deflections  of  two  cantilevers:  a  glass  microneedle  whose 
flexural  stiffness  was  determined  separately  and  an  experimental  spicule.  Made  from 
a  fiber  of  fiberglas,  the  microneedle  was  10  yum  in  diameter  and  1  mm  long  and  was 
fixed  to  the  end  of  a  3  mm  diameter  glass  rod.  The  flexural  stiffness  of  the  needle 
was  determined  by  calculating  /  from  dimensions  and  E  from  equations  of  bending 
for  a  simply  supported  beam  (Gordon,  1978).  Known  weights  (0.2  and  0.5  mg)  were 
hung  on  the  needle  and  vertical  deflection,  measured  in  ^m,  was  photographed  with 
a  horizontally  oriented  photomicroscope.  E  for  the  needle  is  60  GN/m2. 

For  measurement  of  spicule  stiffness,  a  cantilevered  spicule  was  fixed  over  the 
edge  of  a  microscope  slide  with  Eastman  910  cement.  The  needle  and  spicule  were 
aligned  horizontally  in  the  focal  plane  under  a  photomicroscope,  and  the  stage  was 
moved  so  that  the  needle  bent  the  spicule.  Multiple  exposure  photos  were  taken 
because  they  increase  the  accuracy  in  measuring  deflection  of  the  needle  (Fig.  1  ). 
The  photographs  were  analyzed  by  superimposing  the  undeflected  spicule  over  the 
deflected  spicule,  and  then  measuring  the  length  of  both  cantilevers  to  point  of 
contact  and  the  distance  from  this  point  to  the  identical  point  on  each  undeflected 
cantilever  (see  Fig.  1).  The  force  exerted  on  the  deflected  spicule  is  equal  to  that 
exerted  on  the  deflected  needle,  so  flexural  stiffness  was  determined  by  solving  can- 
tilever bending  equations  (Gordon,  1978)  as  follows. 

F    =  3fF  T  Y        VT    3  =  F   =  1>(F  I  Y       VI   3 

*n         -HJ-jn1n  *•  nmax//  ^n  *  s         JvJ-^s1s  l  smax//  J-'s 

where 

Fn  =  force  exerted  by  the  needle  of  the  spicule  (Units:  N) 
Fs  =  force  exerted  by  the  spicule  on  the  needle  (N) 
En  =  modulus  of  the  glass  (N/m2) 

2 


Es  =  modulus  of  the  spicule  (N/m2) 

4 


In  =  second  moment  of  area  of  the  needle  (m4) 
Is  =  second  moment  of  area  of  the  spicule  (m4) 
=  deflection  of  the  needle  where  it  contacts  the  spicule  (m) 
=  deflection  of  the  spicule  where  it  contacts  the  needle  (m) 
Ln  =  length  of  the  needle  to  point  of  contact  with  spicule  (m) 
Ls  =  length  of  the  spicule  to  point  of  contact  with  needle  (m) 


266  RICHARD  B.  EMLET 


FIGURE  1.  Stiffness  measuring  technique,  a  double  exposure.  The  spicule  was  moved  to  contact 
and  bend  the  stationary  needle,  and  the  first  exposure  was  taken.  Then  the  spicule  was  moved  free  of 
the  needle,  and  a  second  exposure  was  taken.  The  image  of  the  undeflected  spicule  was  then  drawn  onto 
the  photo.  Dimensions  were  taken  from  the  photographs.  Black  lines  show  the  length  of  the  cantilever 
needle,  Ln,  and  spicule,  Ls.  Yn  and  Ys  are  the  distances  of  deflection  of  the  needle  and  spicule  respectively. 
See  text  for  further  explanation.  Scale  (white  line):  0.1  mm. 

This  equation  can  be  rewritten  in  the  following  way: 

EsIs  =  (Yn/Ys).(Ls/Ln)3-(EnIn) 

Only  bends  where  Yn/Ln  and  Ys/Ls  ;S  10%  were  used  because  this  equation  is  not 
accurate  for  larger  deflections  where  shear  in  the  material  becomes  increasingly 
important  (Faupel,  1964).  No  attempt  was  made  to  measure  breaking  strength. 

For  simple  spicules  which  are  circular  in  cross-section,  /  =:  1/4  ?rr4  with  r  =  ra- 
dius of  the  spicule.  For  the  fenestrated  spicules,  /  : :  3/4  Trr4  +  3/2  vrrd2,  with  r 
=  radius  of  the  element  rods  and  d  ==  radius  of  the  spicule.  The  amount  (volume) 
of  skeletal  material  in  larval  spicules  was  also  estimated  with  dimensions  taken  from 
SEM  photos.  I  treat  the  fenestrated  spicules  as  three  parallel  circular  cylinders  joined 
by  cross-ties. 

Other  mechanical  calculations  will  be  introduced  as  needed  in  the  discussion. 
They  are  taken  from  Wainwright  et  al.  (1976)  and  Faupel  (1964),  and  may  be  found 
in  most  general  mechanical  engineering  texts. 

RESULTS 

The  spicules  of  echinoplutei  reared  during  this  study  are  500  to  800  ^m  long. 
Simple  spicules  range  in  diameter  from  2  to  4  ^m.  Each  of  the  three  elements  of 
fenestrated  spicules  are  1.5  to  2.5  nm  in  diameter  and  the  whole  spicule  is  5  to  10 
/j.m  in  diameter  (Fig.  2).  Fenestrated  spicules  contain  about  twice  as  much  material 
as  simple  ones  of  similar  length.  In  a  fenestrated  spicule  the  material  is  located 
farther  from  the  bending  axis,  so  these  spicules  should  be  stiffer  than  simple  spicules. 
An  empirical  measure  of  stiffness  rather  than  calculation  of  /  is  used  for  three 
reasons.  For  fenestrated  spicules  the  tapering  width  and  irregular  spacing  of  cross- 
ties  make  accurate  calculation  of  /  difficult.  These  spicules  are  stiffest  at  the  base 
where  the  spicule  enters  the  body  region  of  the  larva.  There  is  also  a  slight  twist  in 
the  member  elements  of  the  fenestrated  spicule  of  Dendraster  (Fig.  2f,  g).  This  60° 
rotation  is  in  the  same  direction  for  all  four  of  the  fenestrated  spicules  in  a  larva. 


ECHINODERM  SKELETAL  MECHANICS  267 

Fenestrated  spicules  are  about  three  times  stiffer  than  simple  spicules  (Fig.  3; 
Mann-Whitney  U  test,  P  <  0.00 1 ).  The  mean  stiffness  measured  for  fenestrated  is 
14.1  X  10~13  ±  2.2  X  10~13  s.e.  and  mean  stiffness  for  simple  spicules  is  3.8  X  10  13 
±0.6  X  10~1?  s.e.  Nm2.  I  report  only  two  data  points  for  the  simple  spicules  of 
Dendraster,  but  these  fall  in  the  same  range  for  simple  spicules  of  S.  franciscanus. 
The  large  variation  in  El  of  the  fenestrated  spicules  (Fig.  3)  is  probably  due  to  a 
large  variation  of  /. 

The  calculated  Young's  modulus  of  calcite  is  36.3  GN/m2  ±  2.9  GN/m2  s.e. 
(n  =  4)  in  the  simple  spicules  which  were  straight  and  had  a  constant  diameter.  The 
E  calculated  for  one  fenestrated  spicule  is  48.9  GN/m2.  No  other  values  of  E  for 
fenestrated  spicules  were  determined  because  of  the  difficulty  of  accurately  calcu- 
lating /. 

Figure  4  shows  some  of  the  calcitic  structures  found  in  Psolus  chitonoides.  These 
structures  are  typical  of  those  found  throughout  adult  echinoderms. 

DISCUSSION 
The  material 

In  this  study  Young's  modulus  (E)  of  echinoderm  calcite  is  determined  to  be 
36.6  GN/m2  (s.e.  ==  2.9,  n  ==  4).  This  value  is  lower  than  all  previous  reports  except 
one.  Burkhardt  and  Ma'rkel  (1980)  give  values  for  E  in  diadematid  spines  as  69.4 
and  52. 1  GN/m2  for  dry  and  wet  spines,  respectively.  Currey  (pg.  167  in  Wainwright 
et  ai,  1967)  gives  values  of  74  and  9.7  GN/m2  in  spines  and  plates  of  echinoids, 
respectively.  Differences  may  be  due  to  methodological  difficulties  of  measuring  E 
and  /  in  previous  studies.  All  of  these  studies  including  the  present  one  calculate 
E  from  EI/I  where  /  is  estimated  from  cross-sections  through  the  structure  and  is 
exclusive  of  voids  in  the  material.  Determination  of/  can  be  difficult  especially  for 
structures  which  have  a  complex  distribution  of  material  around  a  bending  axis  as 
in  echinoderm  stereom,  the  adult  skeletal  plate  structure.  It  is  possible  that  E  values 
previously  reported  differ  from  what  is  found  here  because  the  /  was  inaccurately 
calculated.  When  /  is  calculated  from  dimensions  on  a  photograph,  the  E  is  greater 
for  a  fenestrated  spicule  than  that  for  a  simple  spicule.  This  is  due  to  an  underes- 
timate of  /,  probably  due  to  difficulties  of  evaluating  7  at  cross-ties.  It  is  not  likely 
that  fenestrated  spicules  are  made  from  a  different  calcitic  material.  The  simple  and 
fenestrated  spicules  in  Dendraster  grow  out  of  the  same  triradiate  spicule,  and  simple 
spicules  have  approximately  the  same  E  and  /  as  simple  spicules  from  S. 
franciscanus. 

As  a  material,  echinoderm  calcite  should  no  longer  be  considered  similar  to 
inorganic  calcite.  The  single  crystal  construction  suggested  by  optical  behavior  (e.g., 
Donnay  and  Pawson,  1969)  is  more  apparent  than  real.  Several  authors  (Travis, 
1970;  Pearse  and  Pearse,  1975;  Okazaki  and  Inoue,  1976;  Urakami  et  al.,  1980; 
O'Neill,  1981)  provide  evidence  for  an  oriented  microcrystalline  construction.  In 
addition,  all  of  the  reported  values  for  Young's  modulus  are  two  to  four  times  lower 
than  that  expected  for  inorganic  calcite  (137  GN/m2,  Bhimasenachar,  1945).  These 
two  differences  suggest  that  there  is  an  organic  matrix  in  echinoderm  calcite. 

The  mechanical  properties  of  a  crystalline  material  may  vary  with  the  orientation 
of  the  crystal.  The  modulus  of  inorganic  calcite  is  137  GN/m2  in  the  direction  of 
the  C-axis  but  is  as  low  as  34.2  GN/m2  in  the  other  directions  (Bhimasenachar, 
1945).  Okazaki  and  Inoue  (1976)  confirmed  that  the  C-axis  in  most  larval  spicules 
is  in  the  long  axis  of  the  spicule.  Raup  (1966)  reports  the  same  for  the  orientation 
in  spines,  but  reports  that  the  C-axis  may  be  perpendicular  or  tangential  in  echinoid 


268 


RICHARD  B.  EMLET 


ECHINODERM  SKELETAL  MECHANICS 


269 


Ill 


in 
in 

UJ 


25  - 


20- 


E?    «1 


in 


x 

UJ 


E    10- 

z 


5- 


X 
* 


X 
X 

I 


X 
X 


X 
X 


SIMPLE 
S.f. 


SIMPLE 
D.e. 


F  E  NESTR 
D.e. 


SKELETAL     ROD     TYPE 

FIGURE  3.  Flexural  stiffness  (Newtons  x  meter2  X  10~13)  of  simple  and  fenestrated  spicules.  Mean 
El  of  simple  spicules:  3.8  X  1(T13  N/m2,  s.e.  0.6.  Mean  El  of  fenestrated  spicules:  14.1  X  10  '3,  s.e.  2.2. 
The  stiffnesses  of  the  two  spicule  types  are  significantly  different,  Mann-Whitney  U  test,  P  <  0.001.  S.f., 
Stronglyocentrotusfranciscanus;  D.e.,  Dendraster  excentricus. 

plates.  The  orientation  of  the  C-axis  along  the  long  axis  of  these  structures  means 
that  they  are  stiffer  than  they  would  be  if  the  C-axis  were  in  any  other  orientation. 
With  the  possible  exception  of  the  value  for  the  plate,  the  lower  stiffness  of  echi- 
noderm  calcite  cannot  be  attributed  to  varying  C-axis  orientation. 

Magnesium  replaces  up  to  1 6%  of  the  calcium  in  echinoderm  calcite,  but  the 
reasons  for  the  variation  in  magnesium  content  remain  obscure  (Chave,  1954;  We- 
ber, 1969).  It  is  not  clear  how  magnesium  content  will  affect  the  modulus.  Increasing 
magnesium  content  increases  the  hardness  of  calcite  (Wainwright  et  al,  1976)  and 
therefore  will  probably  increase  stiffness. 

A  porous  microstructure  would  explain  the  reduced  stiffness.  Okazaki  and  Inoue 
(1976)  showed  a  high  magnification  SEM  photo  suggesting  a  porous  surface  on 
carefully  isolated  spicules.  Observation  under  high  magnification  of  the  spicules 
isolated  by  my  own  techniques  never  revealed  that  apparent  texture.  An  empirical 
formula  (by  Mackenzie,  pg.  157  in  Wainwright  et  al.,  1976)  for  change  in  modulus 
in  a  porous  ceramic  predicts  that  a  50%  volume  of  pores  is  necessary  to  give  a  75% 
reduction  in  modulus,  equivalent  to  the  E  reported  here.  Therefore,  a  porous  mi- 
crostructure probably  cannot  account  for  the  reduced  stiffness  of  echinoderm  calcite. 

An  organic  matrix  and  composite  construction  would  also  reduce  the  stiffness 
of  the  calcitic  tissue.  Though  the  collagen  connecting  the  calcitic  plates  may  con- 
taminate some  samples  (Klein  and  Currey,  1970;  Travis,  1970),  there  is  growing 
evidence  for  an  organic  matrix  in  echinoderm  calcite  (Klein  and  Currey,  1970; 

FIGURE  2.  Simple  and  fenestrated  calcareous  spicules  isolated  from  echinoplutei.  (A)  Simple  "half 
skeleton"  of  two  week  old  S.  franciscanus.  (B,  C)  Early  and  later  stages  of  the  fenestrated  "half  skeleton" 
of  D.  excentricus.  In  (C)  the  skeleton  is  modified  and  allows  articulation  of  the  fenestrated  post-oral  rod. 
A,  B,  C,  scale  100  ^m.  (D,  E)  Higher  magnification  of  the  simple  and  fenestrated  spicules.  Note  the 
smooth  surface  of  the  calcite.  D,  E  scale:  10  nm.  (F,  G)  Fenestrated  spicules  of  D.  excentricus.  (F)  Note 
the  taper  and  irregular  spacing  of  cross-ties  in  fenestration.  Scale:  100  ^m.  (G)  Same  spicule  as  (F),  note 
the  twist  in  the  parallel  elements  of  the  fenestrated  part.  Scale:  10 


270 


RICHARD  B.  EMLET 


FIGURE  4.  Plates  and  stereom  of  the  holothurian  Psolus  chitonoides.  (A)  Flat  plate.  Scale:  100 
(B)  A  plate  that  is  becoming  a  laminated  structure  with  the  addition  of  a  new  layer.  Scale:  100  t*m.  (C) 
Labyrinthic  stereom  structure  in  the  form  of  a  block.  Scale:  100  nm.  (D)  Higher  magnification  of  a 
stereom  surface.  Scale:  10 


Travis,  1970;  Pucci-Minafra  et  al.,  1972;  Pearse  and  Pearse,  1975;  Okazaki  and 
Inoue,  1976).  Okazaki  and  Inoue  (1976)  give  an  organic  content  for  the  larval 
spicules  of  about  1%  by  weight.  Klein  and  Currey  ( 1 970)  give  a  value  of  0.3%  protein 
by  weight  (about  1%  by  volume)  which  is  close  to  the  0.36%  for  protein  in  larval 
spicules  given  by  Okazaki  and  Inoue  (1976). 

Evidence  for  a  highly  oriented  microcrystalline  structure  in  echinoderm  calcite 
is  also  increasing.  Polarized  light  and  X-ray  diffraction  studies  (Raup,  1966;  Donnay 
and  Pawson,  1969;  Nissen,  1969)  suggest  that  echinoderm  calcite  is  a  single  crystal, 
but  these  studies  cannot  distinguish  between  a  single  crystal  and  a  highly  ordered 
microcrystalline  construction  where  all  the  microcrystals  have  the  same  C-axis  ori- 
entation. Fracture  studies  do  not  show  cleavage  planes  expected  of  inorganic  crys- 
talline calcite  (Raup,  1966;  Nichols  and  Currey,  1968;  Nissen,  1969;  Okazaki  and 
Inoue,  1976),  and  several  authors  show  fractures  (Pearse  and  Pearse,  1975;  O'Neill, 
1981)  or  etching  (Okazaki  and  Inoue,  1976)  which  suggest  concentric  laminated 
ordering  of  microcrystals.  Recent  studies  by  O'Neill  suggest  that  when  echinoderm 


ECHINODERM  SKELETAL  MECHANICS  27 1 

calcite  is  stressed  in  tension  the  microcrystals  creep,  or  move  with  respect  to  one 
another.  Currey  (1965)  loaded  echinoid  spines  in  bending  and  found  no  creep  after 
26  h.  But,  as  he  states,  the  spines  were  from  dried  specimens,  which  may  have 
prevented  creep  from  occurring. 

If,  in  fact,  this  calcite  tissue  is  a  highly  ordered  "inorganic  polycrystalline  ag- 
gregate" (Travis,  1970)  bound  in  a  very  small  amount  of  organic  matrix  (1  to  2% 
by  volume),  then  mechanically  its  behavior  can  be  treated  as  a  composite  material. 
In  this  treatment  the  microcrystals  are  analogous  to  short  fibers  and  the  organic 
material  is  the  matrix  which  binds  them.  The  modulus,  Ec,  of  the  composite,  mod- 
eled as  a  series  of  layers  of  fibers  and  matrix  can  be  predicted  as  follows: 

1/EC  =  Vf/Ef+Vm/Em 
where 

Vf  =  volume  fraction  of  fibers  =  99% 
Ef  =  modulus  of  fibers  =  137  GN/m2 
Vm  =  volume  fraction  of  matrix  =  1% 
Em  =  modulus  of  matrix  ==  0.6  GN/m2 

andVf+Vm=  1 

(Currey,  pg.  145  in  Wainwright  el  al,  1976.  The  value  of  the  modulus  for  the  matrix 
is  that  of  human  tendon  and  is  meant  to  be  an  approximation  to  the  collagen-like 
component  of  matrix.) 

By  this  formula  Ec  is  evaluated  to  be  4 1 .9  GN/m2  which  is  one  standard  deviation 
higher  than  the  value  of  36.3  GN/m2  determined  in  this  study.  Therefore,  it  may 
be  reasonable  to  treat  the  material,  echinoderm  calcite,  as  a  special  kind  of  composite 
with  a  high  fiber  content.  Although  this  formula  is  used  to  model  composites  whose 
components  are  arranged  in  series  (Reuss  model),  there  is  no  evidence  that  com- 
ponents in  echinoderm  calcite  are  physically  arranged  in  this  way.  The  formula 
merely  predicts  this  composite's  behavior.  Echinoderm  calcite  has  been  called  a 
composite  by  Weber  et  al.  ( 1 969),  but  the  present  work  is  the  first  to  describe  its 
mechanical  behavior  as  a  composite  material. 

Biological  implications 

Comparison  of  the  mechanical  properties  of  echinoderm  calcite  with  that  of 
inorganic  calcite  reveals  the  biological  advantages  in  composite  construction.  A 
spicule  of  composite  construction  should  be  effectively  stronger  than  one  constructed 
from  a  single  inorganic  crystal.  In  theory  the  inorganic  calcite  should  have  a  higher 
fracture  stress  (greater  force  per  unit  area  at  failure),  but  in  practice  tiny  cracks  and 
surface  flaws  set  the  upper  limit  to  fracture  stress  (Wainwright  et  al.,  1976).  A 
composite  construction  of  many  tiny  crystallite  'fibers'  may  reduce  the  possibility 
of  this  common  cause  of  failure  in  brittle  materials  if  cracks  are  not  propagated 
through  the  material  when  a  single  or  a  few  fibers  break  (Wainwright  et  al,  1976; 
Gordon,  1978). 

Brittle  materials  are  usually  weaker  in  tension  than  compression.  This  restricts 
the  usefulness  of  such  a  material  to  sustaining  compressive  loads.  The  composite 
construction  should  increase  the  tensile  strength  over  that  of  inorganic  calcite.  Frac- 
tures caused  by  rapid  loading  usually  do  not  show  inorganic  fracture  planes,  which 
would  require  the  lowest  work  of  fracture.  In  a  composite  material,  in  which  the 
modulus  of  the  fibers  and  the  viscosity  of  the  matrix  are  high,  rapid  loading  should 
crack  through  matrix  and  fiber,  but  under  low  and  even  stress,  the  matrix  would 


272  RICHARD  B.  EMLET 

be  expected  to  shear.  O'Neill's  ( 1 98 1 )  pictures  of  microcrystals  in  prestressed  fracture 
support  this  prediction  of  material  behavior. 

The  larval  spicules 

Arm  rods  of  echinoderm  larvae  are  the  simplest  echinoderm  skeletons  and, 
therefore,  are  a  good  starting  place  for  the  analysis  of  mechanical  properties  at  the 
structural  level.  If  E  is  the  same  for  all  echinoderm  calcite,  then  comparison  of 
stiffness  for  different  structures  can  be  made  through  their  /  values.  (Compare 
/  =  3.9  X  10~23  m4  for  fenestrated  spicules  with  /  :  1.2  X  10~23  m4  for  simple 
spicules.)  While  the  /  of  fenestrated  spicules  is  approximately  three  times  as  much, 
they  contain  only  twice  as  much  calcite  as  simple  spicules.  If  the  same  volume  of 
material  that  is  in  a  fenestrated  spicule  were  arranged  in  a  simple  structure  around 
the  bending  axis,  its  /  value  would  be  about  twice  that  of  the  simple  spicule.  Fen- 
estration  gives  the  spicule  an  increased  stiffness  per  amount  of  material.  Further, 
a  solid  spicule  constructed  with  the  same  dimensions  as  a  fenestrated  spicule  would 
increase  the  stiffness  by  an  order  of  magnitude  over  the  simple  spicule  but  would 
also  require  about  six  times  the  amount  of  material.  The  use  of  less  material  must 
be  important  in  a  planktonic  larva  which  has  to  overcome  gravity  to  stay  afloat. 

Other  benefits  of  fenestration  can  be  appreciated  by  looking  again  at  the  structure 
(Fig.  2e,  f,  g).  Three  parallel  rods  are  the  minimum  above  one  which  give  almost 
even  stiffness  around  a  central  bending  axis.  Two  parallel  rods  will  not  provide  an 
even  distribution  of  /.  The  left-handed  twist  of  about  60°  in  the  parallel  elements 
reduces  stress  along  any  rod  when  the  spicule  is  bent  in  a  certain  direction.  This 
slight  twist,  in  the  same  direction  for  all  fenestrated  spicules  in  Dendraster  larvae, 
probably  reflects  the  construction  pattern  or  orientation  of  the  organic  matrix.  This 
pattern  cannot  be  the  result  of  net  torque  on  the  arm  due  to  swimming  currents, 
because  the  mirror  image  pairs  would  have  opposite  coiling  twists  since  the  currents 
are  subject  to  bilateral  symmetry. 

Fenestration  increases  stiffness  in  torsion  about  a  central  axis.  Here  cross-ties 
increase  the  J  value  (second  polar  moment  of  area),  which  is  a  measure  of  the 
geometric  distribution  of  material  around  a  twisting  axis  and  is  analogous  to  the  / 
value.  Fenestration  also  increases  the  resistance  to  buckling  since  it  is  proportional 
to  flexural  stiffness.  Functions  of  the  larval  skeleton  will  be  discussed  in  more  detail 
in  a  later  paper. 

The  adult  skeleton:  porous  plates  and  stereom 

The  mechanics  of  the  unusual  structure  of  the  adult  skeletal  plates,  called  stereom 
(Fig.  4c,d),  have  been  largely  uninvestigated.  Nichols  and  Currey  (1968)  suggest 
that  the  porous  structure  may  strengthen  echinoderm  calcite  since  small  cracks  stop 
when  they  run  into  a  hole  and  also  point  out  that  this  construction  allows  access 
to  surfaces  for  repair.  As  in  the  composite  construction,  the  porous  structure  should 
reduce  the  difference  between  strength  in  tension  and  strength  in  compression.  Cur- 
rey (1975)  compared  the  crushing  strength  of  echinoderm  stereom  with  that  of 
mollusc  shell  and  found  it  comparable  to  moderately  strong  mollusc  shell  on  a  unit 
weight  basis.  The  crushing  strength  for  echinoderm  stereom  is  50-100  MN/m2 
(Wainwright  et  al.,  1976).  However,  I  calculated  a  stress  of  120  MN/m2  at  14%  tip 
deflection  in  an  unbroken  spicule  during  a  bending  trial  (see  Wainwright  et  al., 
1976,  pg.  248,  for  formula  of  tensile  stress  in  bending). 

Like  fenestrated  spicules,  the  porous  calcite  structures  of  adults  have  the  benefits 
of  increased  bending  and  torsional  stiffness  and  buckling  strength,  when  compared 


ECHINODERM  SKELETAL  MECHANICS  273 

on  a  unit  weight  basis  with  solid  structures.  For  structures  with  the  same  general 
shape  and  composed  of  the  same  weight  of  material,  stiffness  will  increase  faster 
than  strength  with  increasing  porosity.  This  is  because  stiffness  is  proportional  to 
/  which  is  proportional  to  r4  and  strength  is  proportional  to  //r  or  r3.  /  will  be  greater 
for  complex  stereom  than  for  solid  structures  because  material  is  separated  in  space 
around  the  bending  axis  (larger  r).  The  largest  /  occurs  if  the  material  is  distributed 
in  an  annulus  about  the  central  axis.  This  means  that  while  there  are  no  great 
differences  in  the  strength  to  weight  ratios  of  echinoderm  stereom  and  mollusc  shell, 
there  are  differences  in  the  stiffness  or  buckling  strength  to  weight  ratios,  with  echi- 
noderm skeleton  being  greater  in  both. 

Mechanical  properties  may  vary  within  and  between  skeletal  blocks  because  of 
different  stereom  structure.  Smith  (1980)  demonstrated  a  range  of  variation  in  pore 
density  and  pattern  in  the  stereom  structure  of  echinoids  and  described  ten  distinct 
stereom  types.  Macurda  et  al.  (1978)  described  four  of  these  types  for  recent  crinoids. 
Figure  4  shows  three  of  these  types  also  found  in  the  holothurian,  Psolus  chitonoides. 
Reoccurring  stereom  fabrics  suggest  the  possibility  of  mechanical  differences,  but 
no  work  has  demonstrated  this.  A  more  quantitative  analysis  is  needed  that  will 
demonstrate  how,  for  a  given  amount  of  material,  stiffness  and  strength  are  influ- 
enced by  porosity.  This  analysis  may  be  done  by  comparing  the  different  stereom 
structures  by  calculating  Fs  and  cross-sectional  areas  of  material  in  structures  with 
the  different  stereom  types.  Carter  and  Hayes  (1976)  showed  that  different  types  of 
bone  tissue  can  be  treated  similarly  in  mechanical  testing,  and  that  variation  in 
compressive  strength  in  bone  of  different  tissue  morphology  can  be  described  as  a 
function  of  its  relative  density  and  the  compressive  strength  of  compact  bone.  Similar 
studies  on  echinoderm  stereom  should  lead  to  development  of  formulae  which 
describe  strength  or  stiffness  as  a  function  of  density  and  stereom  type. 

The  skeletal  system 

The  organization  of  these  plates  into  functional  skeletons  for  organs  and  organ- 
isms is  highly  varied.  Skeletal  blocks  with  different  mechanical  properties  are  ar- 
ranged and  interconnected  more  or  less  tightly  with  collagen  fibers  (Hyman,  1955) 
and  often  articulated  with  muscle.  The  nature  of  formation  of  these  optical  crys- 
talline blocks  inside  a  syncitium  (Okazaki  and  Inoue,  1976;  Loeper  and  Pearse, 
1981)  may  account  for  the  small  degree  of  variation  (mineral  content)  in  the  com- 
posite material  echinoderm  calcite.  Structural  differences  may  be  viewed  as  the 
method  of  varying  mechanical  properties  of  calcite  materials.  Smith  (1980)  reported 
that  galleried  stereom  is  always  associated  with  long  bundles  of  collagen  fibers. 
Macurda  et  al.  (1978)  found  characteristic  spines  on  the  labyrinthic  stereom  where 
muscles  attach.  One  of  the  intensions  of  Smith's  (1980)  study  was  to  correlate 
stereom  type  with  soft  tissue  type,  but  perhaps  it  may  be  more  appropriate  to  cor- 
relate stereom  type  with  mechanical  operation. 

The  porous  structure  of  echinoderm  calcite  increases  its  flexural  and  torsional 
stiffness,  buckling  strength,  and  possibly  its  tensile  strength  on  a  unit  weight  basis 
over  that  of  a  solid  construction.  A  composite  and  porous  construction  may  allow 
wider  application  of  this  element  in  skeletons  than  just  carrying  compressive  loads. 
Eylers  (1976)  describes  the  distribution  of  forces  in  the  skeleton  of  an  asteroid  during 
the  opening  of  bivalve  prey.  The  ossicles  joined  along  the  aboral  surface  by  collagen 
and  muscle  are  in  tension,  and  ambulacral  ossicles  experience  bending,  torsion,  and 
compression.  Tensile  forces  also  occur  in  the  arms  of  suspension  feeding  crinoids 
and  ophiuroids,  spines  of  echinoids,  imbricate  plate  systems,  and  most  other  ex- 


274  RICHARD  B.  EMLET 

amples  of  echinoderm  skeleton.  The  mechanical  behavior  of  an  intact  adult  skeleton 
should  then  be  analyzed  as  an  interaction  between  composite  blocks,  collagen  con- 
nective tissues,  and  muscle.  The  mechanical  diversity  of  echinoderm  skeletal  or- 
ganization may  also  be  attributed  to  material  and  structural  properties  of  echinoderm 
calcite. 

ACKNOWLEDGMENTS 

I  am  especially  grateful  to  R.  Strathmann  for  suggestions  and  criticisms  on  all 
stages  of  this  work.  I  thank  A.  O.  D.  Willows,  Director  of  Friday  Harbor  Labs,  for 
making  facilities  available  for  this  work.  Discussions  with  B.  Best,  M.  Denny,  M. 
LaBarbera,  M.  Koehl,  S.  Smiley  and  others  helped  me  clarify  some  of  the  ideas 
presented  here,  but  all  mistakes  are  my  own.  I  am  also  grateful  to  S.  A.  Wainwright 
whose  comments  have  improved  this  manuscript.  This  research  was  supported  by 
NSF  Grant  number  8008310  to  R.  Strathmann. 


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MECHANISM  OF  THE  EXCITATION-CONTRACTION  UNCOUPLING  OF 
FROG  SKELETAL  MUSCLE  BY  FORMAMIDE 


GLADYS  ESCALONA  DE  MOTTAt,  DAVID  S.  SMITH*,  MARILYN  CAYER**, 

AND  JOSE  DEL  CASTILLOft 

t 

•f  College  of  Pharmacy  and  ^Laboratory  of  Neurobiology.  University  of  Puerto  Rico,  Medical  Sciences 

Campus,  Rio  Piedras.  PR,  * Department  of  Zoology,  Oxford  University.  England,  and 

**Papanicolaou  Cancer  Research  Institute,  Miami,  FL 

ABSTRACT 

The  contractility  of  guinea  pig  ileum  and  frog  skeletal  muscle  is  inhibited  in 
solutions  containing  0.4  to  2.5  M  formamide  (FMD).  Contrary  to  mammalian  vis- 
ceral muscle,  this  blocking  action  is  not  reversed  when  frog  muscles  are  transferred 
back  to  isotonic  Ringer's  after  FMD  treatment.  Under  these  conditions  the  water 
content  of  the  skeletal  muscles  is  markedly  increased  and  electronmicrographs  show 
a  swelling  of  the  transverse  tubules.  These  changes  are  not  observed  when  frog  muscles 
are  transferred  to  ethylene  glycol  solutions  that  are  isosmotic  with  the  FMD  containing 
Ringer's  solution.  In  addition,  over  50%  of  the  contractility  is  recovered  in  these 
muscles.  These  observations  provide  direct  evidence  of  the  occurrence  of  an  osmotic 
shock  in  frog  muscles  transferred  from  FMD  solutions  to  isotonic  Ringer's.  It  is 
concluded  that  the  resulting  alterations  in  the  triad  structure  and  function  are  re- 
sponsible for  the  irreversibility  of  the  FMD  uncoupling  action  in  these  muscles. 

INTRODUCTION 

Formamide  (FMD),  added  to  isotonic  saline  at  concentrations  between  0.4  and 
2.5  M,  produces  an  immediate  and  completely  reversible  inhibition  of  the  shortening 
of  mammalian  visceral  muscle  (Cordoba  et  #/.,  1968)  and  blocks  irreversibly  with 
a  slower  time  course  the  contractility  of  frog  skeletal  muscle  (del  Castillo  and  Es- 
calona  de  Motta,  1978).  The  skeletal  muscle  fibers  blocked  by  FMD  retain  their 
electrical  and  chemical  excitability  properties  and  are  able  to  respond  with  fast  local 
twitches  to  the  electrophoretic  injection  of  Ca2+  (Escalona  de  Motta  and  del  Castillo, 
unpublished  observations).  In  addition,  caffeine  still  induces  slow  sustained  con- 
tractures  in  these  muscles  (Escalona  de  Motta  et  al.,  1982).  These  observations 
suggest  that  the  effects  of  FMD  on  frog  skeletal  muscle  are  exerted  on  the  coupling 
between  excitation  and  contraction.  In  this  sense,  FMD  may  be  classified,  together 
with  glycerol  and  ethylene  glycol,  as  an  excitation-contraction  (E-C)  uncoup- 
ling agent. 

However,  the  uncoupling  action  of  glycerol  and  ethylene  glycol  does  not  occur 
until  the  muscles  are  suddenly  transferred  back  to  isotonic  Ringer's,  inducing  an 
osmotic  shock  that  disrupts  the  tubules  of  the  T-system.  (Eisenberg  and  Gage,  1 967). 
With  FMD,  muscles  lose  their  contractility  while  still  immersed  in  the  hypertonic 

Received  13  May  1982;  accepted  15  July  1982. 

Address  reprint  requests  to:  Laboratory  of  Neurobiology,  201  Blvd.  del  Valle,  San  Juan,  PR  00901, 
USA. 

Abbreviations:  E-C,  excitation-contraction;  EG.  ethylene  glycol;  FMD,  formamide;  SR,  sarcoplasmic 
reticulum. 

276 


E-C  UNCOUPLING  BY  FORMAMIDE  277 

solution,  indicating  that  an  osmotic  shock  is  not  essential  for  the  uncoupling  action 
exerted  by  FMD  (del  Castillo  and  Escalona  de  Motta,  1978).  The  present  work 
investigates  the  possible  osmotic  effects  of  FMD  solutions  on  frog  sartorius  muscles, 
measuring  changes  in  the  water  content  of  these  muscles  and  examining  the  ultra- 
structure  of  the  muscle  fibers  under  various  conditions. 

MATERIALS  AND  METHODS 

Preparations.  Sartorius  muscles  of  small  (2")  frogs  (Rana  pipiens)  were  dissected, 
with  or  without  the  sciatic  nerve  attached,  and  pinned  to  a  layer  of  Sylgard  (Dow 
Corning)  at  the  bottom  of  a  small  Petri  dish.  Contractility  was  determined  visually 
by  observing  the  twitches  induced  by  stimulating  the  muscle  directly  or  via  the 
attached  motor  nerve  using  a  pair  of  platinum  electrodes.  All  the  applied  stimuli 
were  square  pulses  of  1  ms  duration  and  supramaximal  strength.  In  experiments 
where  the  tension  developed  was  measured,  the  muscles  were  tied  at  both  ends  with 
silk  threads  and  placed  in  a  vertical  bath  containing  Ringer's  solution.  The  muscle, 
attached  to  the  chamber  by  one  end,  was  connected  by  the  other  end  to  a  Grass  FT 
103  isometric  transducer  connected  to  a  chart  recorder. 

Phvsiological  solutions.  The  mammalian  Krebs-Ringer's  solution  employed  had 
the  following  ionic  composition  (mM):  Na+,  1 18;  K+,  4.6;  Ca2+,  2;  Mg2+,  0.9;  Cl", 
1 17;  HCO3  ,  17.6;  SO42  ,  0.9;  H2PO4  ,  0.9.  A  mixture  of  98%  02  and  2%  CO2  was 
bubbled  continuously  through  the  solution,  which  had  a  pH  of  7.3  after  equilibra- 
tion. Glucose  (5  mM)  was  added  to  this  solution.  Direct  measurement  of  osmolarity 
with  a  cryosmometer  gave  values  ranging  between  295  and  305  mOsm/liter. 

The  frog  Ringer's  solution  used  contained  the  following  ionic  concentrations 
(mM):  Na+,  1 17;  K+,  2.1;  Ca2+,  1.87,  all  as  chloride  salts.  The  pH  of  this  solution 
was  adjusted  to  7.2  with  5  mM  TES  (N-tris  hydroxymethyl  methyl-2-  aminoethane- 
sulfonic  acid)  and  NaOH.  The  osmolarity  of  this  solution,  determined  with  an 
Advanced  Instruments  osmometer,  ranged  from  242  to  250  mOsm/liter.  FMD  or 
ethylene  glycol  (EG)  was  added  to  these  solutions  in  the  concentrations  further 
indicated. 

Measurement  of  water  content  in  muscle.  The  muscles  were  weighed  at  regular 
intervals  before  and  after  they  were  immersed  in  Ringer's  solution  to  which  different 
amounts  of  FMD  had  been  added.  Extreme  care  was  taken  in  handling  the  muscles 
to  ensure  reproducibility  of  the  results.  After  each  series,  the  muscle  was  dried  over 
a  desiccant,  until  there  was  no  further  change  in  weight.  The  water  content  of  each 
muscle  was  calculated  by  subtracting  the  dry  weight  from  the  original  wet  weight. 

Ultrastructural  experiments.  Muscles  which  were  in  normal  Ringer's  saline  were 
fixed  in  a  solution  containing  2%  glutaraldehyde  in  0.05  M  cacodylate  buffer  (pH 
7.4)  with  4%  sucrose  for  24  hours  at  4°C,  and  the  solution  was  changed  several 
times  during  the  first  hours. 

Other  muscles  were  transferred  directly  from  a  2.0  M  FMD  solution  in  Ringer's 
to  a  fixative  like  the  one  above,  but  also  containing  2.0  M  FMD. 

Finally,  a  third  group  of  muscles  was  sequentially  transferred  from  1 .0  M  FMD 
to  isosmotic  ethylene  glycol  and  then  to  the  basic  fixative  solution  to  which  1 .0  M 
of  ethylene  glycol  had  been  added. 

All  specimens  were  treated  subsequently  with  1%  osmium  tetroxide  in  0.05  M 
cacodylate  buffer  for  1  hour  at  4°C,  dehydrated  in  an  ethanol  series,  and  embedded 
in  Araldite.  Sections  were  cut  with  glass  or  diamond  knives  on  an  LKB  Ultratome 
III,  stained  with  ethanolic  uranyl  acetate  and  lead  citrate,  and  examined  in  a  Philips 
EM  400  and  200. 


278 


G.  ESCALONA  DE  MOTTA  ET  AL. 


RESULTS 

Osmotic  effects  of  high  FMD  concentrations.  FMD  is  a  highly  permeant  solute, 
as  shown  by  the  fact  that  the  water  content  of  ileal  strips  placed  in  2  M  FMD  in 
Krebs-Ringer's  solution  show  no  appreciable  change  in  weight  after  20  min.  In 
addition,  as  shown  in  Figure  1 ,  no  significant  changes  in  water  content  could  be 
detected  when  these  ileal  strips  were  transferred  back  to  normal  Krebs-Ringer's. 

The  movement  of  FMD  across  the  membrane  systems  of  skeletal  muscle  is 
slightly  more  restricted.  Indeed,  frog  sartorius  muscles  exhibit  a  small  ( 1 5%)  decrease 
in  total  weight  when  immersed  in  2  M  FMD-Ringer's,  and  almost  double  their  water 
content  when  transferred  back  to  normal  Ringer's  (see  Figure  2).  This  last  obser- 
vation suggests  that  FMD  does  not  leave  the  skeletal  muscle  fibers  as  easily  as  it 
goes  into  them,  thus  favoring  the  occurrence  of  an  osmotic  shock  similar  to  that 
brought  about  in  skeletal  muscles  exposed  to  hypertonic  glycerol  (Gage  and  Eisen- 
berg,  1969). 

Ultrastructure  of  muscles  equilibrated  in  hypertonic  FMD.  To  determine  whether 
the  block  of  contraction  which  occurs  while  muscles  are  still  immersed  in  the  FMD 
solution  could  be  correlated  with  ultrastructural  changes  caused  by  hypertonicity, 
two  muscles  blocked  after  1 8  min  in  2.0  M  FMD  were  transferred  to  a  fixing  solution 
that  also  contained  2.0  M  FMD.  Post-fixation  and  further  processing  of  these  spe- 
cimens was  then  done  as  described  in  Materials  and  Methods.  Figures  3  and  4  show 
that  the  myofibrils  and  associated  sarcoplasmic  reticulum  (SR),  as  well  as  the  mem- 
branes of  the  tubular  system  (T-system),  of  these  muscles  are  normal  in  appearance 
and  do  not  present  any  obvious  morphological  features  that  may  be  associated  with 
a  loss  of  contractility. 

Structural  changes  in  FMD-blocked  muscles  transferred  to  normal  Ringer's.  Two 
muscles  blocked  after  18  min  in  2  M  FMD-Ringer's  were  transferred  to  isotonic 
Ringer's  and  washed  extensively  for  30  min  before  fixation. 

Figure  5  and  6  illustrate  the  marked  changes  in  ultrastructure  observed.  There 
are  large  numbers  of  "lacunae"  irregularly  distributed  throughout  the  fields  but 


no-, 


NR 


1OO 


9 

I 


4)      90 


Time    (min) 

FIGURE  1.  The  water  content  of  guinea  pig  ileum  strips  does  not  change  appreciably  upon  exposure 
to  2  M  FMD  and  after  transfer  to  normal  Ringer's  (NR).  Arrow  indicates  the  change  from  FMD  or  NR. 
Top  curve  is  the  control  muscle  maintained  in  NR  throughout  the  experiment.  See  Materials  and  Methods 
for  experimental  procedure. 


E-C  UNCOUPLING  BY  FORMAMIDE 


279 


140 


120 


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Time   (min) 

FIGURE  2.  Frog  sartorius  muscles  equilibrated  in  2  M  FMD  increase  markedly  their  water  content 
when  brought  back  to  normal  Ringer's  (arrow).  Muscle  marked  NR  was  maintained  in  normal  Ringer's 
throughout  the  experiment.  Experimental  procedure  is  explained  in  Materials  and  Methods. 

always  lying  at  the  level  of  the  Z  bands  of  the  myofibrils.  In  these  regions,  the  T- 
tubules  are  swollen  but  maintain  their  close  association  with  the  membrane  of  the 
terminal  cisternae  of  the  SR. 

Recovery  of  contractility  in  FMD-blocked  muscles  transferred  to  isosmotic  EG. 
To  test  whether  the  observed  osmotic  shock  was  per  se  the  cause  of  the  irreversibility 
of  the  uncoupling  action  of  FMD  on  skeletal  muscles,  we  performed  experiments 
avoiding  the  occurrence  of  drastic  osmotic  changes.  Pairs  of  sartorius  muscles  were 
exposed  to  a  1.0  M  FMD  solution  (1,190  mOsm)  for  a  period  sufficiently  long  to 
produce  a  complete  loss  of  contractility  at  this  lower  FMD  concentration.  These 
muscles  were  then  transferred  to  a  1  M  EG  solution  ( 1 ,2 10  mOsm).  Ethylene  glycol 
has  been  employed  in  similar  concentrations  to  produce  E-C  uncoupling  of  frog 
muscles  by  an  osmotic  shock  (Sevcick  and  Narahashi,  1972),  but,  by  itself  and  in 
contrast  to  what  we  have  shown  with  FMD,  it  does  not  impair  muscle  contraction 
(Caputo,  1968). 

Both  the  water  content  of  the  muscles  and  their  contractility  in  response  to  direct 
electrical  stimulation  were  determined  in  these  preparations.  Figure  7  shows  that 
the  water  content  of  these  muscles  decreases  slightly  after  transfer  to  the  EG  solution, 
indicating  that  this  alcohol  does  not  penetrate  freely  into  the  muscle  fibers.  Figure 
8  illustrates  the  time  course  of  the  blockade  of  a  muscle  exposed  to  1  M  FMD  and 
the  partial  recovery  of  contractility  after  24  min  in  EG.  As  emphasized  in  the 
discussion,  the  reduced  force  of  contraction  of  muscle  treated  by  FMD  may  be  due 
to  the  occlusion  of  a  fraction  of  the  T-tubules. 

Structure  of  muscles  fixed  after  successive  exposure  to  FMD  and  EG.  Figure  9 
shows  a  survey  field  of  a  sartorius  muscle  fiber  exposed  sequentially  to  1.0  M  FMD 
and  isosmotic  solution  prior  to  fixation  with  a  glutaraldehyde  solution  that  also 
contained  1 .0  M  EG.  The  appearance  of  the  relaxed  myofibrils  and  the  associated 


280  G.  ESCALONA  DE  MOTTA  ET  AL. 


FIGURE  3.  Frog  sartorius  muscle  fixed  in  glutaraldehyde  in  presence  of  2  A/formamide  (see  Materials 
and  Methods).  The  myofibrils  (in  this  instance  contracted)  and  associated  SR  and  T-system  membranes 
are  essentially  normal  in  appearance.  Arrows  indicate  triads  situated,  as  is  characteristic  of  this  muscle, 
at  the  Z  band  level.  Scale  bar:  1 


E-C  UNCOUPLING  BY  FORMAMIDE 


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FIGURE  4.  Material  prepared  as  in  Figure  3,  illustrating  details  of  the  triad  configurations.  This 
field  includes  an  extensive  T-tubule  profile  (T),  flanked  by  terminal  cisternae  (SR).  The  width  of  the  triad 
gap  and  the  spacing  of  the  foot  or  pillar  processes  stemming  from  the  SR  are  as  in  conventionally  fixed 
material.  The  components  of  the  triad  are  further  illustrated  in  the  inset,  in  which  the  T-tubule  is 
transversely  sectioned:  note  the  normal  disposition  of  the  foot  processes.  Scale  bars:  0.25  ^tm;  inset, 
0.1 


282 


G.  ESCALONA  DE  MOTTA  ET  AL. 


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FIGURE.  5.  Frog  sartorius  muscle  sequentially  soaked  in  2  M  formamide,  washed  in  Ringer's  and 
fixed  in  glutaraldehyde.  (See  Materials  and  Methods  for  times  of  treatment).  In  this  survey  field,  the 
obvious  abnormality  is  the  presence  of  'lacunae1  (arrows),  irregularly  scattered  through  the  material,  of 
varying  size  but  invariably  lying  at  the  Z  band  levels.  These  are  further  illustrated  in  Figure  6.  Scale  bar: 
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FIGURE  6.  Material  fixed  as  in  Figure  5,  at  higher  magnification.  Portions  of  three  triads  are  included 
in  tangential  section  in  this  field,  encircling  the  underlying  myofibril.  These  are  'normal'  in  appearance 
for  part  of  their  course  (arrows)  but  on  the  right  of  the  field  (T)  the  medial  T-tubules  are  swollen  to 
varying  degrees,  providing  the  Z-level  'lacunae'  seen  in  Figure  5.  As  in  this  instance,  the  swelling  is  often 
irregular  along  the  course  of  an  individual  tubule,  occurring  primarily  between  or  at  the  periphery  of  the 
circumfibrillar  triads.  However,  as  shown  in  the  inset,  the  swelling  sometimes  affects  the  triad  itself.  This 
micrograph  includes  a  severely  abnormal  triad:  the  T-tubule  is  grossly  swollen,  but  retains  its  original 
association  with  the  terminal  cisternae  (SR).  Scale  bars:  0.5  nm;  inset,  0.25 

283 


2S4 


G.  ESCALONA  DE  MOTTA  ET  AL. 


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FIGURE  7.  Changes  in  the  water  content  of  two  frog  sartorius  muscles  equilibrated  with  1.0  A/ 
FMD  and  transferred  to  either  normal  Ringer's  (FMD-NR)  or  1.0  A/  EG  (FMD-EG).  The  curve  marked 
NR  was  obtained  with  a  muscle  maintained  in  normal  Ringer's  throughout  the  experiment.  Arrow 
indicates  the  moment  in  which  the  muscles  were  changed  from  the  FMD  solution.  See  Materials  and 
Methods  for  an  explanation  of  the  experimental  procedure. 

triadic  junctions  is  essentially  normal.  In  particular  the  tubular  swellings  obvious 
in  Figures  5  and  6  are  absent. 

DISCUSSION 

The  results  of  the  experiments  described  above  demonstrate  the  occurrence  of 
an  osmotic  shock  in  frog  sartorius  muscles  transferred  from  FMD  solutions  to  nor- 
mal isotonic  Ringer's.  The  fact  that  in  any  given  field  of  the  electron  micrographs 
obtained  from  these  muscles  not  all  the  T-tubules  are  swollen  suggests  that  only 
some  of  these  links  with  the  plasma  membrane  remain  open  under  these  conditions. 
Quantitative  analysis  of  this  effect  is  rendered  very  difficult  by  the  irregularity  of  the 
swelling,  which  occurs  primarily  between  the  fibrils  or  at  the  edge  of  the  circum- 
fibrillar  triads. 

The  open  tubules  may  be  those  that  become  swollen  by  the  inflowing  isotonic 
fluid.  The  non-swollen  tubules,  which  have  an  essentially  normal  appearance,  are 
possibly  those  that  have  sealed  off  during  the  FMD  treatment  becoming,  effectively, 


FIGURE  8.  Time  course  of  the  blockade  of  contractility  of  a  frog  sartorius  muscle  immersed  in 
1 .0  M  FMD  and  its  partial  recovery  upon  transfer  to  1 .0  M  EG  (*).  a  records  the  control  twitch  tension 
developed  in  response  to  direct  electrical  stimuli  at  a  frequency  of  1  Hz.  At  arrow,  FMD  was  added  to 
the  bath  solution,  c,  d,  e  and  /  were  recorded  3,  6,  12,  and  24  min  after.  /  illustrates  the  blockade  after 
32  min  in  FMD.  h.  i  and  j  were  recorded  after  placing  the  muscle  in  EG  for  6,  12,  and  24  min.  Vertical 
calibration,  0.5  g;  recording  times,  1  min. 


E-C  UNCOUPLING  BY  FORMAMIDE 


285 


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FIGURE  9.  A  survey  field  of  sartorius  muscle  soaked  sequentially  in  1  M  formamide  and  isosmotic 
EG  prior  to  fixation  in  glutaraldehyde.  The  appearance  of  the  myofibrils  (in  this  instance,  relaxed)  and 
associated  triad  junctions  (arrows)  is  essentially  normal;  in  particular,  the  T-tubule  swelling  seen  in  Figures 
5  and  6  is  absent.  Scale  bar:  1  /urn. 


286  G.  ESCALONA  DE  MOTTA  ET  AL. 

intracellular  structures.  In  this  respect,  they  would  be  similar  to  the  terminal  cisternae 
of  the  sarcoplasmic  reticulum,  which  do  not  appear  to  be  noticeably  altered  or 
swollen  in  any  of  the  micrographs.  Conversely,  it  is  possible  that  swelling  occurs 
only  in  sealed  tubules,  but  this  does  not  affect  the  functional  interpretation. 

Thus,  the  irreversibility  of  the  uncoupling  action  of  FMD  on  frog  skeletal  muscle 
transferred  to  normal  Ringer's  may  be  attributed  both  to  the  sealing  of  many  of  the 
T-tubules  and  to  the  swelling,  and  consequent  loss  of  function,  of  others.  In  both 
instances,  the  normal  triadic  structure  and/or  function  would  be  altered,  resulting 
in  complete  loss  of  E-C  coupling. 

This  conclusion  is  supported  by  the  two  above-mentioned  observations:  1)  In 
guinea  pig  ileum,  a  smooth  muscle  that  lacks  a  tubular  system,  there  is  no  obvious 
osmotic  change  when  FMD-equilibrated  strips  are  placed  back  in  isotonic  Krebs- 
Ringer's  where  the  block  of  contractility  is  completely  reversed.  2)  When  an  osmotic 
shock  is  avoided,  by  transferring  frog  sartorius  muscles  to  isosmotic  EG  solutions, 
there  is  a  slow  and  partial  recuperation  of  contractility  suggesting  that  enough  of 
the  T-tubules  remain  open  to  permit  effective  E-C  coupling. 

We  have  observed  changes  in  the  after-potentials  of  spikes  induced  in  muscles 
transferred  to  normal  Ringer's  after  FMD-blockade  which  further  suggest  the  oc- 
currence of  tubular  disruption  (Escalona  de  Motta  et  al.,  1982).  This  led  us  to 
propose  that  FMD  exerts  two  separate  effects  on  muscle  contractility:  a)  a  direct 
reversible  inhibition,  similar  to  that  observed  in  guinea  pig  ileum,  probably  related 
to  an  interference  with  the  activating  action  of  Ca2+  on  the  contractile  machinery; 
and  b)  an  irreversible  effect  occurring  only  when  skeletal  muscles  equilibrated  in 
hypertonic  FMD  solutions  are  suddenly  brought  back  to  normal  saline. 

The  present  observations  emphasize  that  FMD  has  a  direct  inhibitory  action  on 
the  E-C  coupling  process  that  reverses  slowly  when  the  amide  is  removed  from  the 
preparation,  avoiding  drastic  osmotic  changes.  FMD  must  then  be  included  among 
the  permeant  solutes  suitable  for  uncoupling  excitation  from  contraction.  However, 
as  reported  earlier  (Escalona  de  Motta  et  #/.,  1982),  compared  to  other  agents  in 
this  category,  FMD  treatment  is  far  more  gentle  and  better  preserves  the  electrical 
parameters  of  the  muscle  fibers. 

ACKNOWLEDGMENTS 

This  work  was  supported  by  USPHS  grants  RR-08102,  NS- 10447,  NS- 14938, 
and  NS-07464.  We  wish  to  thank  Ms.  Minerva  Rodriguez  Miranda  for  typing  the 
manuscript. 

LITERATURE  CITED 

CAPUTO,  C.  1968.  Volume  and  twitch  tension  changes  in  single  muscle  fibers  in  hypertonic  solutions. 

J.  Gen.  Physio/.  52:  793-809. 
CORDOBA,  F.,  S.  SCHOOF,  S.  VELEZ,  ANDJ.  DEL  CASTILLO.  1968.  Inhibitory  action  of  formamide  on 

smooth  muscle  contraction.  Life  Sci.  7:  897-903. 

DEL  CASTILLO,  J.,  AND  G.  ESCALONA  DE  MOTTA.  1978.  A  new  method  for  excitation-contraction  un- 
coupling in  frog  skeletal  muscle.  J.  Cell  Biol.  78:  782-784. 
EISENBERG,  R.  S.,  AND  P.  W.  GAGE.  1967.  Frog  skeletal  muscle  fibers:  changes  in  electrical  properties 

after  disruption  of  transverse  tubular  system.  Science  158:  1700. 
ESCALONA  DE  MOTTA,  G.,  F.  CORDOBA,  M.  DE  LEON,  ANDJ.  DEL  CASTILLO.  1982.  Inhibitory  action 

of  high  formamide  concentrations  on  excitation-contraction  coupling  in  skeletal  muscle,  J. 

Neurosci.  Res.  1:  163-178. 
GAGE,  P.  W.,  AND  R.  S.  EISENBERG.  1969.  Action  potentials,  after-potentials  and  excitation-contraction 

coupling  in  frog  sartorius  fibers  without  transverse  tubules.  J.  Gen.  Physiol.  53:  298-310. 
SEVCICK,  C.,  AND  T.  NARAHASHI.  1972.  Electrical  properties  and  excitation-contraction  coupling  in 

skeletal  muscle  treated  with  ethylene  glycol.  J.  Gen.  Physiol.  60:  221-236. 


Reference:  Biol.  Bull.  163:  287-300.  (October,  1982) 


RHYTHMS  IN  LARVAL  RELEASE  BY  AN  ESTUARINE  CRAB 
(RHITHROPANOPEUS  HARRISII} 

R.  B.  FORWARD,  JR.,  K.  LOHMANN,  AND  T.  W.  CRONIN1 

Duke  University  Marine  Laboratory,  Beaufort,  NC  28516,  and 
Zoology  Department,  Duke  University.  Durham,  NC  27706 

ABSTRACT 

Ovigerous  females  of  the  crab  Rhithropanopeus  harrisii  were  collected  subtidally, 
and  their  rhythms  in  larval  release  monitored  under  constant  conditions  in  the 
laboratory.  Larvae  from  a  single  crab  are  generally  released  as  a  burst  lasting  less 
than  1 5  minutes.  Larval  release  by  crabs  from  an  estuary  lacking  regular  tides  mainly 
occurs  in  the  2-h  interval  after  sunset  and  is  not  related  to  coastal  tides,  which 
suggests  a  circadian  rhythm.  This  rhythm  can  be  entrained  on  an  altered  light-dark 
cycle.  Larval  release  by  crabs  from  an  estuary  with  semi-diurnal  tides  begins  at  high 
tides  and  continues  for  2  hours,  suggesting  a  circatidal  rhythm.  Significantly  more 
releases  occur  during  the  night.  Crabs  from  the  estuary  without  regular  tides  change 
from  a  circadian  to  a  circatidal  rhythm  after  being  in  the  estuary  with  semi-diurnal 
tides.  Alternatively,  crabs  from  the  estuary  with  semi-diurnal  tides  change  to  a 
circadian  rhythm  when  exposed  to  a  light-dark  cycle  and  non-tidal  conditions  in 
the  laboratory.  Thus  R.  harrisii  has  both  circadian  and  circatidal  rhythms  in  larval 
release  with  the  expressed  rhythm  dependent  upon  prior  environmental  conditions. 
Nighttime  release  may  reduce  predation,  while  release  at  high  tide  may  minimize 
larval  exposure  to  stressful,  low  salinity  water. 

INTRODUCTION 

Rhythms  in  reproductive  activity  and  larval  release  are  common  among  crus- 
taceans. Timing  may  be  related  to  lunar  phase,  time  of  day,  and/or  phase  of  the 
tide.  Semilunar  cycles  are  known  for  semi-terrestrial  crabs  (Clifford,  1962;  Warner, 
1967;  Henning,  1975;  Klassen,  1975;  Saigusa  and  Hidaka,  1978;  Seiple,  1979;  Sai- 
gusa,  1981),  intertidal  fiddler  crabs  (von  Hagen,  1970;  Zucker,  1976,  1978;  Christy, 
1978;  Wheeler,  1978),  and  subtidal  stomatopods  (Reaka,  1976).  For  the  lobsters 
Nephrops  norvegicus  (Moller  and  Branford,  1979),  Homarus  gammarus  (Ennis, 
1973;  Branford,  1978),  and  H.  americanus  (Ennis,  1975),  larval  release  in  the  lab- 
oratory occurs  shortly  after  dusk  on  a  series  of  consecutive  nights;  no  lunar  or 
semilunar  rhythm  has  been  reported.  H.  americanus  occasionally  releases  larvae 
during  the  day  (Ennis,  1975). 

Detailed  laboratory  studies  of  estuarine  intertidal  fiddler  crabs  indicate  that  fe- 
males release  their  larvae  within  several  hours  after  the  time  of  the  nocturnal  high 
tide  (DeCoursey,  1979;  Bergin,  1981).  As  implied  by  DeCoursey  (1979),  precisely 
timed  larval  release  may  not  be  restricted  to  intertidal  fiddler  crabs  but  could  also 
extend  to  other  estuarine  species. 

This  study  was  undertaken  to  examine  larval  release  by  the  estuarine  crab  Rhith- 
ropanopeus harrisii  which  occurs  from  the  very  low  intertidal  zone  into  subtidal 

Received  10  March  1982;  accepted  16  July  1982. 

'  Present  address:  Department  of  Biology,  Yale  University,  New  Haven,  CT  0651 1. 

287 


288  R.  B.  FORWARD  ET  AL. 

areas  (Williams,  1965).  Crabs  from  an  estuary  having  pronounced  semi-diurnal  tides 
were  compared  to  crabs  from  an  estuary  lacking  regular  tides.  Experiments  were 
designed  to  determine  the  presence  of  biological  rhythms  in  larval  release,  the  re- 
lationship of  release  time  to  environmental  cycles,  and  the  ability  of  the  crab  to 
change  its  rhythm  under  different  environmental  conditions. 

MATERIALS  AND  METHODS 

Ovigerous  female  Rhithropanopeus  harrisii  (Gould)  were  collected  from  two 
coastal  estuaries  in  North  Carolina,  the  Neuse  River  (estuary  A)  and  the  Newport 
River  (estuary  B).  Tides  in  estuary  A  are  aperiodic  (Roelofs  and  Bumpus,  1953). 
Physical  factors  such  as  salinity,  water  depth,  and  wave  turbulence  which  usually 
vary  with  the  tides,  vary  instead  with  wind  direction  and  rain.  In  contrast,  estuary 
B  has  regular  semi-diurnal  tides  and  periodic  variation  in  tide-related  hydrography 
(Cronin,  1982). 

Crabs  were  obtained  in  wire  mesh  traps.  In  estuary  A  traps  were  placed  at  a 
depth  of  about  1  m  on  a  gradually  sloping  bottom.  Traps  in  estuary  B  were  placed 
in  an  area  having  a  relatively  uniform  depth  of  3-4  m  at  high  tide. 

Ovigerous  crabs  were  collected  during  the  day,  separated  in  the  laboratory  ac- 
cording to  embryo  development  (based  on  yolk  consumption  and  eye  development), 
and  placed  in  20-cm  diameter  finger  bowls  containing  water  of  the  same  salinity 
as  at  the  collection  site.  The  proper  salinity  was  obtained  by  diluting  sea  water 
(filtered  to  remove  particles  larger  than  5  yum)  with  distilled  water.  Crabs  were  either 
placed  under  constant  conditions  of  temperature,  salinity,  and  light,  or  entrained 
to  a  new  LD  cycle  in  an  environmental  chamber  (Sherer  Gillett  Co.,  Model  CE1 
4-4).  None  were  fed. 

Larval  release  usually  occurs  during  a  specific  interval  in  the  LD  or  tidal  cycle. 
The  time  was  determined  by  intensively  monitoring  larval  release  over  a  designated 
5-h  sampling  interval  within  the  LD  or  tidal  cycle.  During  this  5-h  period,  crabs 
were  transferred  every  15  min  to  a  new  7.9-cm  diameter  finger  bowl.  At  the  end 
of  the  sampling  period,  the  crabs  were  placed  in  10.4-cm  diameter  finger  bowls,  and 
if  eggs  remained,  the  procedure  was  repeated  at  the  next  monitoring  time,  either 
7.4  or  19  h  later  (see  below). 

The  number  of  larvae  released  within  each  1 5-min  interval  and  between  sam- 
pling intervals  was  recorded.  Most  larvae  are  released  within  1 5  min,  though  a  few 
commonly  appeared  in  the  intervals  immediately  preceding  and  following  the  peak. 
The  mean  time  was  calculated  by  multiplying  the  number  of  larvae  released  per  15 
min  by  that  interval,  taking  the  sum  of  these  products  over  all  intervals,  and  dividing 
this  sum  by  the  total  number  of  larvae.  In  this  way  a  single  1 5-min  interval  was 
designated  as  the  time  of  larval  release.  If  a  crab  released  the  majority  of  its  larvae 
during  the  period  between  the  5-h  sampling  periods,  release  was  designated  as  oc- 
curring at  "other  times."  About  12%  of  the  crabs  released  bursts  of  larvae  during 
two  consecutive  sampling  periods.  Using  the  above  procedure  a  mean  time  was 
calculated  for  each  release. 

A  chi-square  test  for  goodness  of  fit  was  used  to  determine  whether  the  number 
of  releases  during  the  intensive  sampling  time  differed  from  an  expected  uniform 
rate  throughout  the  solar  day  or  over  an  entire  tidal  cycle.  For  these  tests,  the  solar 
day  included  a  sampling  time  and  the  preceding  19  h,  while  a  12.4-h  tidal  cycle 
encompassed  the  sampling  time  and  the  preceding  7.4  h.  For  crabs  from  estuary  B, 
a  chi-square  test  was  used  to  determine  any  preference  for  releasing  at  day  or  night- 
time high  tides.  A  Kolmogorov-Smirnov  goodness  of  fit  test  was  used  to  determine 


CRAB  LARVAL  RELEASE  RHYTHMS  289 

if  releasing  was  nonuniform  throughout  the  intensive  sampling  time.  Finally,  linear 
regression  analysis  was  used  to  estimate  the  period  length  of  the  rhythms  by  the 
population.  For  crabs,  which  release  a  burst  of  larvae  during  two  consecutive  sam- 
pling intervals,  only  the  time  of  the  first  burst  was  used  in  this  analysis.  In  this  way 
each  crab  only  contributed  one  time  to  the  data.  Larval  release  was  monitored  in 
five  situations.  The  specific  procedures  for  each  situation  are  described  in  the  next 
section. 

RESULTS 

Estuary  A:  crabs  from  natural  conditions 

In  preliminary  experiments  larval  release  by  crabs  from  estuary  A  was  monitored 
at  2-h  intervals  under  constant  laboratory  conditions  for  3  days.  Releases  began  just 
after  sunset  and  continued  for  several  hours.  Releasing  could  be  related  to  time  of 
day  or  perhaps  to  tides,  even  though  tides  are  considered  aperiodic  at  the  collec- 
tion site. 

To  distinguish  between  these  possibilities  crabs  were  collected  at  weekly  intervals 
for  one  month  (May  16  to  June  13,  1981).  The  tidal  phase  at  dusk  on  the  nearby 
coast  alternated  weekly  between  spring  high  tides  and  neap  low  tides.  After  collection 
and  embryo  staging,  all  crabs  were  maintained  under  room  lights  until  the  time  of 
normal  sunset  when  they  were  placed  in  constant  low  level  light  (photographic 
safelight  containing  a  15-W  bulb  and  fitted  with  a  Kodak  OA  filter;  wavelength 
maximum  ==  573  nm,  half  band  pass  =  37  nm,  intensity  =  1.2  X  10~2  W/m2),  and 
temperature  (28  ±  1  °C).  A  crab  remained  under  constant  conditions  until  it  released 
its  larvae  or  until  6  nights  had  elapsed  and  the  experiment  was  terminated.  Beginning 
1  h  before  the  time  of  the  first  sunset,  all  crabs  with  advanced  embryos  were  trans- 
ferred through  the  series  of  finger  bowls.  Other  crabs  were  tested  as  their  embryos 
matured. 

At  both  collection  times,  each  crab  had  embryos  at  one  stage  of  development. 
However  within  the  collected  crabs,  embryo  development  was  not  uniform,  as  all 
stages  were  observed.  The  number  of  crabs  that  released  larvae  in  the  laboratory 
within  6  nights  of  collection  was  similar  at  both  collection  times  (high  tide  collection, 
n  =  133;  low  tide  collection,  n  =  1 10).  These  results  suggest  there  is  no  lunar  or 
semi-lunar  cycle  in  larval  release. 

During  spring  high  tides  significantly  more  releases  occurred  during  the  sampling 
time  on  nights  1-5  (Fig.  1  A;  nights  1-4,  P  <  0.005;  night  5,  P  <  0.025)  than  expected 
if  releasing  occurred  uniformly  throughout  the  solar  day.  Furthermore,  releasing 
was  not  uniformly  distributed  within  the  5-h  sampling  intervals  on  nights  1-4  and 
6  (nights  1  and  6,  P  <  0.01;  night  3,  P  <  0.02;  nights  2  and  4,  P  <  0.05).  Therefore 
larval  release  by  the  population  occurred  during  a  relatively  short  time  within  the 
4-h  interval  after  sunset. 

Similarly,  when  low  tides  occurred  during  the  evening  the  total  number  of  re- 
leases during  the  intensive  sampling  time  was  greater  than  expected  (Fig.  IB;  nights 
1-6,  P  <  0.005).  Furthermore,  releasing  within  the  sampling  time  was  nonuniform 
on  nights  1,  3,  4,  5,  and  6  (night  4,  P  <  0.01;  night  6,  P  <  0.02;  nights  1,  3,  and 
5,  P  <  0.05).  Again  larvae  were  released  mainly  within  several  hours  after  sunset. 

There  was  no  significant  difference  in  the  distribution  of  release  times  on  specific 
nights  during  evening  high  and  low  tides  (Mann- Whitney  U  test),  (i.e.,  comparison 
of  nights  1,  nights  2,  etc.).  To  further  compare  the  two  situations,  a  regression  was 
determined  for  the  relationship  of  release  time  and  night  in  constant  conditions. 
Night  1  was  excluded  because  release  times  may  be  influenced  by  initial  adjustments 


290 


R.  B.  FORWARD  ET  AL. 


N   I  Coastal  evening 

n=2l  Low  tide 


-I     Sunset         .1 


•  4        Other        Sunset          ,1 
times 

Time  (h) 


«4     Other 
times 


FIGURE  1.  Number  of  crabs  releasing  larvae  (ordinate)  at  times  (abscissa)  relative  to  sunset  in 
estuary  A.  Crabs  were  under  constant  conditions  in  the  laboratory  and  releasing  was  monitored  during 
a  5-h  interval  on  successive  nights  (N)  when  spring  high  tides  (A)  or  neap  low  tides  (B)  occurred  during 
the  evening  on  the  nearest  coast.  On  N 1  in  A  high  tide  occurred  at  about  the  time  of  sunset  while  low 
tide  occurred  at  this  time  in  B.  The  sample  size  (n)  on  each  night  is  shown  and  "other  times"  indicates 
the  number  of  releases  at  times  other  than  the  sampling  time.  For  example  the  releases  shown  on  N2 
for  "other  times"  indicate  those  that  occurred  between  the  end  of  the  first  and  beginning  of  the  second 
sampling  interval.  The  second  release  of  a  crab  is  indicated  by  an  open  histogram. 


to  laboratory  conditions.  The  slopes  of  the  regression  lines  for  releasing  at  both 
evening  high  and  low  tides  were  significantly  different  from  zero  (/-test;  P  <  0.01). 
When  the  two  regressions  were  compared  by  an  analysis  of  covariance,  neither  the 
slopes  nor  the  intercepts  were  significantly  different  (F-test).  These  findings  suggest 
release  time  is  not  related  to  coastal  tides,  and  the  data  in  Figures  1  and  2  were 
therefore  combined  for  the  following  analysis. 

Larval  release  predominantly  occurred  within  a  specific  time  interval  on  con- 
secutive days  in  constant  conditions,  which  suggests  the  crabs  have  an  endogenous 
rhythm.  The  period  of  the  rhythm  of  individual  crabs  can  be  estimated  from  the 
time  between  consecutive  larval  releases.  Nineteen  percent  of  the  crabs  released  on 
two  consecutive  nights.  The  mean  time  between  releases  was  24  h  (SE  =  15  min; 
n  =  25),  when  rounded  off  to  the  nearest  15-min  interval. 

In  addition  the  period  length  of  the  population  rhythm  can  be  estimated  by  a 
regression  analysis  of  the  relationship  between  release  time  and  night  after  placement 
in  constant  conditions  (Fig.  2).  Night  1  was  not  included,  and  only  releases  during 
the  sampling  interval  were  considered.  On  nights  5  and  6  the  number  of  crabs 
releasing  at  times  other  than  the  intensive  sampling  time  increased.  Nevertheless 
these  nights  were  included  because  a  significantly  (P  <  0.005)  greater  number  of 
crabs  released  in  the  5-h  sampling  interval  than  predicted  if  releasing  was  uniform 
over  the  solar  day.  The  slope  of  the  regression  line  (Fig.  2)  is  significantly  different 


CRAB  LARVAL  RELEASE  RHYTHMS 


291 


+4 

+3 

+2 

_c 

1  +' 
f~ 

sunset 


23456 

Night 

FIGURE  2.  Regression  of  time  of  larval  release  (ordinate)  for  the  combined  data  from  Figures  1  and 
2  on  the  consecutive  nights  in  the  laboratory  (abscissa).  The  first  release  by  all  crabs  within  the  intensive 
sampling  interval  was  used  for  the  analysis  while  the  mean  and  standard  deviation  are  shown  on  the 
figure. 


from  zero  (Mest;  P  <  0.001 ),  and  predicts  a  time  of  24  h  23  min  between  consecutive 
releases.  Thus  both  the  time  between  releases  by  a  single  crab  and  the  regression 
analysis  of  the  population  indicate  the  free  running  period  length  is  near  24  h.  This 
implies  the  presence  of  a  circadian  rhythm  in  constant  conditions  in  the  laboratory 
and  a  daily  rhythm  in  nature. 

Estuary  B:  crabs  from  natural  conditions 

Larval  release  by  these  crabs  occurs  near  the  time  of  high  tide  (Cronin  and 
Forward,  1982).  To  determine  the  precise  relationship  between  release  time  and 
tide,  crabs  were  collected  at  weekly  intervals  several  hours  before  daytime  high  tide 
during  July  and  August,  1980  and  198 1.  Since  larval  release  was  monitored  in  crabs 
collected  in  the  same  area  at  the  same  time  of  the  year,  data  from  the  two  years 
were  pooled.  Crabs  were  collected  at  a  depth  where  daylight  probably  is  not  visible 
(see  below);  thus  crabs  were  returned  to  the  laboratory  in  opaque  bottles  and  then 
sorted  according  to  state  of  embryo  development.  The  only  assured  times  the  crabs 
experienced  light  were  the  short  intervals  during  removal  from  the  traps  and  when 
the  embryos  were  staged.  All  crabs  were  then  placed  under  constant  conditions  and 
larval  release  monitored  as  described  previously.  The  first  sampling  time  began  just 
after  staging  of  embryos.  For  this  tide,  crabs  with  the  most  advanced  embryos  were 
monitored  for  4  h  beginning  about  1  h  before  high  tide  in  the  field.  On  the  following 
6  tides,  crabs  with  mature  embryos  were  monitored  from  2  h  before  high  tide  in 
the  field  to  3  h  later. 

Larval  release  by  crabs  from  estuary  B  is  not  uniform  (tides  1-7;  P  <  0.005)  over 
a  complete  tidal  cycle  (Fig.  3).  Ninety-three  percent  of  all  releases  occurred  near 
high  tide  during  the  sampling  time.  Within  this  interval,  releasing  was  distributed 
uniformly  except  for  tides  1  and  2  (P  <  0.01),  when  most  larvae  were  released  in 
the  2-h  interval  after  high  tide. 

Releasing  occurred  near  the  times  of  high  tides,  which  suggests  the  crabs  have 
a  biological  rhythm.  At  the  individual  level,  fifteen  percent  of  the  crabs  (n  =  25) 


292 


R.  B.  FORWARD  ET  AL. 


-I  HT 

Time  (h) 


3  Other 
times 


FIGURE  3.  Number  of  crabs  releasing  larvae  (ordinate)  at  times  relative  to  high  tide  (HT)  in  estuary 
B.  Larval  release  was  monitored  for  a  5-h  period  around  HT  at  the  times  of  successive  high  tides  (T)  in 
the  field  (abscissa).  The  sample  size  (n)  for  each  high  tide  is  shown  and  "other  times"  indicates  the 
number  of  releases  at  times  other  than  the  sampling  interval.  For  example,  the  releases  shown  on  T3  for 
"other  times"  are  the  number  that  occurred  between  the  end  of  the  second  and  beginning  of  the  third 
sampling  interval.  The  second  release  of  a  crab  is  indicated  by  an  open  histogram. 


released  larvae  during  successive  high  tides,  while  about  24  h  elapsed  between  releases 
by  one  crab.  The  mean  time  between  successive  releases  was  12  h  15  min  (SE  =  15 
min)  when  rounded  off  to  the  nearest  15-min  interval.  The  relationship  between 
larval  release  by  the  population  and  tide  (Fig.  4)  is  significant  (/-test;  P  <  0.001). 
Furthermore,  the  slope  of  the  regression  line  indicates  the  time  between  releases  on 
consecutive  tides  is  12  h  12  min.  Multiple  releases  by  a  single  crab  as  well  as  the 
regression  analysis  of  the  population  indicate  that  the  free  running  period  length  is 
around  12  h  15  min.  This  implies  the  presence  of  a  circatidal  rhythm  under  constant 
conditions  in  the  laboratory  and  a  tidal  rhythm  in  nature. 

Although  releases  occurred  on  successive  tides,  it  is  possible  that  there  is  a  day/ 
night  component  in  the  rhythm.  The  average  natural  photoperiod  throughout  the 
experiments  was  14  h  light  and  10  h  dark.  If  releasing  is  independent  of  the  light- 
dark  cycle,  then  the  predicted  frequencies  during  the  day  and  night  sampling  in- 
tervals would  be  58  and  42%,  respectively.  The  observed  frequencies  during  the  day 
and  night  are  48  and  52%.  Releasing  was  not  uniform  during  daytime  and  nighttime 
high  tides  (chi-square  test,  P  <  0.005),  as  a  significantly  greater  amount  occurred 
near  the  time  of  nighttime  high  tides. 


CRAB  LARVAL  RELEASE  RHYTHMS 


293 


HT 


O) 


-2 


4 

Tide 


.     7 


FIGURE  4.  Regression  of  time  (±  SD)  of  larval  release  (ordinate)  on  the  various  tides  (abscissa)  for 
crabs  from  estuary  B.  The  first  release  by  all  crabs  within  the  intensive  sampling  interval  was  used  for 
the  analysis  while  the  mean  and  standard  deviation  are  shown  on  the  figure. 


Estuary  A:  entrainment  to  a  LD  cycle  in  the  laboratory 

Two  methods  were  used  to  determine  if  the  apparent  circadian  rhythm  of  crabs 
from  estuary  A  could  be  entrained  by  a  light-dark  cycle.  In  the  first  method,  crabs 
were  collected  during  the  day  and  those  with  embryos  in  early  stages  of  development 
were  held  at  27.0°C  under  a  14:10  LD  cycle  in  the  environmental  chamber.  The 
light  intensity  during  the  day  phase  was  about  2.0  W/m2  (cool  white  fluorescent 
lamps).  The  length  of  the  photoperiod  was  similar  to  that  in  the  field,  but  the 
beginning  of  the  dark  phase  occurred  6  h  before  sunset.  The  crabs  were  maintained 
under  these  conditions  for  5  days  because  preliminary  experiments  showed  this 
duration  was  sufficient  to  reset  the  timing  of  the  rhythm.  Crabs  were  then  placed 
under  the  same  constant  conditions  as  described  above  for  crabs  from  estuary  A. 
The  method  for  monitoring  release  was  also  similar  except  that  the  sampling  time 
began  1  h  before  the  end  of  the  light  phase.  Crabs  were  monitored  for  3  days. 

The  time  of  larval  release  shifted  with  an  altered  LD  cycle  in  the  laboratory. 
When  the  time  of  "lights  off'  (laboratory  sunset)  occurred  6  h  before  the  normal 
sunset  in  the  field,  the  time  of  releasing  shifted  similarly  after  5  days  of  entrainment 
(Fig.  5).  Clearly,  releasing  was  not  uniform  over  the  day,  since  it  only  occurred 
during  the  sampling  time.  Within  this  interval,  releasing  was  also  not  uniform  on 
all  3  nights  (P  <  0.05).  Larval  release  began  at  the  end  of  the  light  phase  and  con- 
tinued for  about  the  next  1.5  h.  A  regression  analysis  of  the  population  release  times 
was  not  performed  because  crabs  were  monitored  only  over  3  days.  Nevertheless, 
at  the  individual  level  13%  of  the  crabs  (n  =  10)  released  larvae  on  consecutive 
nights.  The  mean  time  between  releases  was  24  h  1 5  min  (SE  =  1 5  min)  when 
rounded  off  to  the  nearest  1 5-min  interval,  which  approximates  the  suggested  free 
running  period  length  of  population  from  the  field  (Fig.  2). 

The  second  method  involved  monitoring  larval  release  by  crabs  that  were  main- 
tained under  summer  conditions  during  the  winter  in  a  laboratory  habitat.  Crabs 


294 


R.  B.  FORWARD  ET  AL. 


FIGURE  5.  Number  of  crabs  from  estuary  A  releasing  larvae  (ordinate)  at  times  (abscissa)  relative 
to  the  end  of  the  light  phase.  Crabs  were  placed  on  a  14:10  LD  cycle  in  the  laboratory  for  5  days  and 
then  maintained  under  constant  conditions.  "Lights  oft""  indicates  the  end  of  the  light  phase  on  the  first 
night  and  "sunset"  is  the  time  of  sunset  in  estuary  A.  Other  symbols,  as  in  Figure  1.  No  releases  occurred 
at  other  times. 


were  held  at  26.0°C  in  9  ppt  sea  water,  subjected  to  a  summer  photoperiod  (15:9 
LD  cycle;  cool  white  fluorescent  lamps  positioned  over  the  tank;  intensity 
:  7.5  W/m2)  and  fed  with  Purina  cat  chow.  Females  became  ovigerous  in  January 
and  breeding  continued  through  the  spring.  The  method  for  monitoring  releasing 
was  identical  to  that  for  the  previous  crabs  on  an  altered  LD  cycle.  However  during 
constant  conditions  the  crabs  were  placed  in  the  environmental  cabinet  (tempera- 
ture, 26°C)  having  low  intensity  red  light  (6.5-W  red  incandescent  lamp;  wavelength 
output  was  greater  than  600  nm;  intensity  about  0.3  W/m2).  Releasing  was  moni- 
tored for  3  days. 

Winter  crabs  also  exhibited  a  rhythm  in  larval  release  that  was  entrained  to  the 
altered  LD  cycle  (Fig.  6A).  Only  4%  of  the  crabs  did  not  release  larvae  in  the  sampling 
time  which  indicates  releasing  was  nonuniform  over  the  solar  day  (P  <  0.005).  On 
all  3  nights  the  release  distribution  was  not  uniform  within  the  sampling  interval 
(P  <  0.0 1 ).  Again  releasing  began  at  the  end  of  the  light  phase  and  continued  for 
about  the  next  1.5  h.  Eleven  percent  of  the  crabs  (n  =  5)  released  larvae  on  con- 
secutive nights,  with  a  mean  time  between  releases  of  23  h  30  min  (SE  =  15  min) 
when  rounded  off  to  the  nearest  1 5-min  interval. 

Estuary  B:  entrainment  to  a  LD  cycle  in  the  laboratory 

To  determine  whether  crabs  from  estuary  B  could  change  from  an  apparent 
circatidal  to  a  circadian  rhythm,  crabs  with  embryos  which  would  hatch  between 
6  and  9  days  after  capture  were  collected  shortly  before  high  tide  and  placed  in  the 
environmental  chamber  under  a  14:10  LD  cycle  (cool  white  fluorescent  lamps  plus 
a  60-W  incandescent  bulb;  intensity  =  9.0  W/m2)  at  27 °C.  In  order  to  separate  daily 
and  tidal  influences,  the  time  of  the  LD  cycle  was  adjusted  so  that  the  dark  phase 


CRAB  LARVAL  RELEASE  RHYTHMS 


295 


_o 

0 

6 


0> 

.0 

E 

- 


Lights 
off 


.2  .3 


*4       Other          Lights          tl 
times  off 

Time  (h) 


3  .4       Other 

times 


FIGURE  6.  Larval  release  by  crabs  from  estuary  A  (A)  and  estuary  B  (B)  that  reproduced  during 
the  winter.  Crabs  were  maintained  on  a  15:9  LD  cycle,  and  releasing  was  monitored  under  constant 
conditions.  "Lights  off"  indicates  the  end  of  the  light  phase  on  the  first  night.  Other  symbols,  as  in 
Figure  1. 


began  at  the  predicted  time  of  daytime  low  tide  in  the  field  6  days  after  collection. 
After  6  or  7  days  crabs  were  placed  under  constant  conditions  similar  to  those  used 
for  the  tidal  rhythm  experiments  with  crabs  from  estuary  B  and  larval  release  was 
monitored  intensively  for  5.5  h,  beginning  1  h  before  the  beginning  of  the  dark 
phase.  Preliminary  experiments  indicated  that  6  days  was  the  minimum  time  nec- 
essary for  the  crabs  to  change  their  rhythm.  Releasing  was  monitored  for  only  3 
days  because  the  total  time  for  embryonic  development  is  about  10  days.  Few  crabs 
still  had  eggs  at  the  end  of  the  experiment. 

Crabs  released  larvae  during  the  1.5-h  interval  after  the  end  of  the  light  phase 
(Fig.  7)  rather  than  during  the  time  following  high  tide  in  the  field.  Releasing  was 
neither  uniform  during  the  solar  day  (P  <  0.05)  nor  during  the  sampling  interval 
for  all  nights  (P  <  0.05).  Eight  percent  of  the  crabs  (n  =  5)  released  larvae  on  con- 
secutive nights.  The  mean  time  between  releases  was  24  h  (SE  =  1 5  min). 

To  further  establish  that  crabs  from  estuary  B  can  develop  a  circadian  rhythm, 
crabs  were  maintained  over  the  winter  in  a  habitat  identical  to  that  used  for  crabs 
from  estuary  A,  and  releasing  was  monitored  using  similar  procedures.  Larval  release 
by  winter  crabs  was  also  related  to  the  LD  cycle  (Fig.  6B),  as  only  1%  of  the  crabs 
did  not  release  larvae  during  the  intensive  sampling  interval  (nonuniform  releasing 
over  the  solar  day,  P  <  0.005).  Releases  were  nonuniform  within  the  sampling  in- 
terval on  all  nights  (P  <  0.05)  with  most  releases  occurring  in  the  1.5-h  interval  after 
the  end  of  the  light  phase.  Twelve  percent  of  the  crabs  (n  =  8)  released  larvae  on 
consecutive  nights.  The  mean  time  between  releases  was  23  h  30  min  (SE  =  1 5  min), 
which  corresponds  to  the  time  observed  for  crabs  from  estuary  A  under  similar 
conditions  (Fig.  6A). 

Estuary  A:  entrainment  to  natural  tidal  conditions 

A  final  experiment  determined  whether  crabs  from  estuary  A  could  change  from 
an  apparent  circadian  to  a  circatidal  rhythm  in  larval  release.  Since  the  environ- 
mental cycles  which  entrain  the  tidal  rhythms  are  unknown,  male  and  nonovigerous 
female  crabs  from  estuary  A  were  placed  in  plastic  boxes  containing  mollusk  shells 
in  traps  at  the  collection  site  in  estuary  B.  Holes  in  each  box  permitted  water  flow 
through  the  box  but  prevented  the  crabs  from  escaping.  Beginning  10  days  after 
crabs  were  translocated  to  estuary  B,  ovigerous  females  were  collected  at  weekly 


296 


R.  B.  FORWARD  ET  AL. 


Other 
times 


FIGURE  7.  Number  of  releases  (ordinate)  at  times  (abscissa)  relative  to  the  end  of  the  light  phase 
for  crabs  from  estuary  B  which  were  placed  on  a  14:10  LD  cycle  in  the  laboratory  for  6  or  7  days  and 
then  maintained  under  constant  conditions.  "Lights  off"  indicates  the  end  of  the  light  phase  on  the  first 
night.  Other  symbols,  as  in  Figure  1 . 


intervals  for  a  month.  The  crabs  were  collected  several  hours  before  daytime  high 
tide,  and  larval  release  measured  as  described  for  crabs  from  estuary  B  (tidal  ex- 
periments). 

When  crabs  were  transferred  from  estuary  A  to  estuary  B,  they  developed  an 
apparent  circatidal  rhythm  (Fig.  8).  Eighty-four  percent  of  all  releases  occurred 
during  the  5-h  sampling  time  around  high  tide  (nonuniform  releasing  over  the  tidal 
cycle,  P<  0.005),  though  releasing  was  uniform  within  this  interval  on  all  tides. 
There  was  no  significant  preference  for  day  or  night  high  tides.  Three  of  the  crabs 
released  larvae  during  consecutive  sampling  times. 

DISCUSSION 

The  crab  Rhithropanopeus  harrisii  shows  rhythms  in  larval  release  that  are  re- 
lated to  environmental  cycles  in  the  habitat  where  it  lives.  At  the  collection  site  in 
estuary  A,  crabs  experience  the  natural  light-dark  cycle  and  a  diel  temperature  cycle 
(unpublished  observations).  Tides  in  this  area  are  aperiodic  (Roelofs  and  Bumpus, 
1953)  even  though  tidal  currents  may  occur  in  the  estuary  (Knowles,  1975).  In  the 
experiments,  larval  releases  of  crabs  from  this  estuary  were  not  related  to  tides  but 
rather  began  at  sunset  and  continued  for  about  2  h  (Fig.  1 ).  The  observed  time  of 
larval  release  does  not  reflect  the  monitoring  regime  or  handling,  since  the  identical 
release  pattern  was  observed  in  preliminary  experiments  when  crabs  were  handled 
every  2  h  for  3  days. 

The  relationship  between  releasing  and  the  time  of  sunset  suggests  the  presence 
of  a  circadian  rhythm.  A  circadian  rhythm  is  normally  defined  as  an  endogenous 
rhythm  which  persists  for  at  least  5-10  cycles  in  a  single  individual  under  constant 


CRAB  LARVAL  RELEASE  RHYTHMS 


297 


Other 
times 


FIGURE  8.    Larval  release  by  crabs  from  estuary  A  that  were  transplanted  to  estuary  B.  Notations 
as  in  Figure  3. 


conditions  and  which  has  a  free  running  period  close  to  but  not  exactly  24  h.  Larval 
release  occurs  either  as  a  single  event  in  one  crab  or,  at  most,  two  events  on  con- 
secutive nights.  Thus  the  criteria  of  persistence  of  the  rhythm  in  a  single  individual 
for  5-10  cycles  cannot  be  fulfilled.  Nevertheless,  the  rhythmic  release  of  larvae  by 
a  population  of  crabs  does  persist  under  constant  conditions  in  the  laboratory  for 
at  least  6  diel  cycles  (Fig.  1).  Also,  the  time  between  consecutive  releases  by  a  single 
crab  and  the  regression  analysis  of  population  release  times  (Fig.  2)  indicate  free 
running  period  lengths  of  nearly  24  h.  These  considerations  suggest  that  there  is  a 
circadian  rhythm  in  larval  release  by  individual  crabs  in  constant  conditions  in  the 
laboratory  which  is  observed  as  rhythmic  releases  by  the  population  within  a  specific 
time  interval  on  successive  days.  Furthermore,  these  results  indicate  that  the  pop- 
ulation has  a  daily  rhythm  in  larval  release  in  nature. 

In  contrast,  crabs  from  an  estuary  with  semi-diurnal  tides  had  the  greatest  num- 
ber of  releases  in  the  2-h  interval  after  high  tide  (Fig.  3).  This  pattern  is  not  due  to 
the  sampling  regime,  since  similar  results  were  obtained  in  preliminary  experiments 
when  the  crabs  were  sampled  every  2  h  for  6  days  (Cronin  and  Forward,  1982). 
These  results  suggest  the  presence  of  a  circatidal  rhythm.  The  rhythmic  release  of 
larvae  by  the  population  persisted  for  seven  tidal  cycles  under  constant  conditions 
and  had  a  free  running  period  of  about  1 2  h  1 5  min  (Fig.  4)  as  did  successive  releases 
from  a  single  crab.  In  nature  the  population  would  have  a  tidal  rhythm  in  larval 
release. 

These  crabs  also  had  a  significantly  greater  number  of  larval  releases  at  night, 
although  a  considerable  number  of  releases  occurred  during  daytime  high  tides.  The 
absence  of  a  strong  preference  for  day  or  night  may  be  due  to  environmental  con- 
ditions. The  crabs  were  collected  in  traps  at  a  depth  of  3-4  m.  Here  they  would  be 
exposed  to  pronounced  tidal  changes  in  current  flow,  depth,  and  salinity  (Cronin, 
1982),  but  it  is  unlikely  that  they  would  sense  a  diel  LD  cycle.  This  prediction  is 


298  R.  B.  FORWARD  ET  AL. 

based  upon  the  rapid  attenuation  of  light  in  the  estuary  and  assumes  that  the  adults 
have  the  same  spectral  (Forward  and  Cronin,  1979)  and  intensity  sensitivity  (Cronin, 
1979)  as  their  larvae.  Since  the  crabs  can  move  from  shallow  to  deep  areas,  we  were 
probably  sampling  crabs  which  had  ;nd  had  not  been  exposed  recently  to  the  natural 
LD  cycle.  This  may  explain  the  weak  preference  for  night  in  our  results. 

The  crabs  change  both  the  time  of  larval  release  and  the  length  of  the  free 
running  period  when  exposed  to  different  environmental  cycles.  Crabs  from  estuary 
A  altered  the  time  of  releasing  when  entrained  to  a  new  LD  cycle  in  the  laboratory 
(Figs.  5  and  6A).  Under  a  LD  cycle  and  nontidal  conditions  in  the  laboratory,  crabs 
from  estuary  B  developed  a  circadian  rhythm  (Figs.  6B  and  7).  All  crabs  (estuaries 
A  and  B)  that  were  entrained  to  a  LD  cycle  in  the  laboratory,  began  larval  release 
just  after  the  end  of  the  light  phase  and  continued  for  about  2  hours.  Releasing 
occurred  at  the  same  time  for  field-captured  animals  (Fig.  1),  which  suggests  the 
LD  cycle  is  the  normal  zeitgeber  in  the  field. 

A  circatidal  rhythm  was  induced  in  crabs  from  estuary  A  by  placing  them  in 
estuary  B  (Fig.  8),  but  the  zeitgeber  remains  unknown.  The  phase  of  the  rhythm 
was  similar  to  that  for  crabs  living  in  estuary  B.  Interestingly,  these  crabs  had  a 
pronounced  daily  rhythm  in  their  original  habitat,  yet  lacked  a  day/night  preference 
after  exposure  to  conditions  in  estuary  B.  The  transported  crabs  were  placed  in 
boxes  at  3-4  m  depth  with  no  possibility  of  movement  to  shallower  depths.  It  is 
unlikely  the  crabs  experienced  a  LD  cycle  at  this  depth,  which  may  explain  the 
absence  of  a  preference  for  larval  release  at  night. 

The  crabs  from  both  estuaries  have  the  capability  of  showing  both  circatidal  and 
circadian  rhythms  after  exposure  to  different  environmental  conditions.  Larval  re- 
lease by  R.  harrisii  is  not  the  only  example  of  this  flexibility.  For  example,  this  was 
also  found  in  activity  rhythms  of  the  crabs  Carcinus  maenas  (Naylor,  1958,  1960) 
and  Uca  tangeri  (Altevogt,  1959)  and  in  vertical  migration  patterns  of  R.  harrisii 
larvae  (Cronin  and  Forward,  1979).  Of  particular  interest,  however,  is  the  time  of 
release  with  respect  to  the  tidal  and  diel  LD  cycles,  since  larval  release  could  be  most 
adaptive  at  a  particular  time  of  day  or  phase  of  the  tide. 

When  not  exposed  to  periodic  tides,  R.  harrisii  shows  a  daily  rhythm  with 
releasing  occurring  primarily  in  the  2-h  interval  after  sunset.  Larval  release  at  night 
is  commonly  observed  in  laboratory'  studies  of  crustaceans  such  as  lobsters  (Pandian, 
1970;Ennis,  1973,  1975;  Branford',  1978;  Moller  and  Branford,  1979),  fiddler  crabs 
(DeCoursey,  1979,  1981;  Bergin,  1981),  and  the  prawn  Macrobrachium  idae  (Pan- 
dian and  Katre,  1972).  Nighttime  release  has  been  observed  in  field  studies  of  fiddler 
crabs  (Hyman,  1922;  Christy,  1978;  DeCoursey,  1981;  Stancyk  and  Christy,  1981), 
pebble  crabs  (Knudsen,  1960),  Cardisoma  guamhumi  (Gifford,  1962),  Aratus  pisoni 
(Warner,  1967),  Birgus  latro  (Reese  and  Kinzie,  1968)  and  various  Sesarma  species 
(Saigusa,  1981).  This  suggests  nocturnal  larval  release  has  a  common  functional 
advantage,  which  is  probably  avoidance  of  predators  on  larvae  and  adults  which 
visually  sight  and  actively  pursue  their  prey  (Ennis,  1975;  Branford,  1978;  De- 
Coursey, 1979;  Bergin,  1981). 

Larval  release  by  R.  harrisii  from  a  tidal  area  occurs  primarily  in  the  2-h  interval 
after  the  time  of  high  tides.  In  other  detailed  studies  of  crustaceans  from  an  estuarine 
tidal  area,  fiddler  crabs  (DeCoursey,  1979,  1981;  Bergin,  1981)  and  Sesarma  sp. 
(Saigusa,  1981)  had  similar  times  of  larval  release.  Since  releases  frequently  occur 
near  high  tide,  this  again  suggests  a  common  functional  advantage. 

In  estuaries,  larvae  encounter  the  problems  of  transport  and  of  survival  in  a 
highly  variable  environment.  By  entering  the  water  column  near  the  time  of  high 
tide,  subsequent  horizontal  movements  are  seaward  as  the  tide  recedes.  This  would 
tend  to  favor  larval  dispersal.  Although  this  explanation  is  appropriate  for  fiddler 


CRAB  LARVAL  RELEASE  RHYTHMS  299 

crabs  (Wheeler,  1978;  Christy,  1978;  Bergin,  1981;  Stancyk  and  Christy,  1981)  and 
Sesarma  sp.  (Saigusa  and  Hidaka,  1978;  Saigusa,  1981),  it  is  unlikely  that  the  time 
of  release  by  R.  harrisii  aids  dispersal,  since  field  studies  (Bousfield,  1955;  Pin- 
schmidt,  1963;  Sandifer,  1973,  1975;  Cronin,  1982)  indicate  that  massive  seaward 
transport  of  larvae  does  not  occur,  and  that  all  larval  stages  are  retained  in  the  area 
of  the  adult  population  (Cronin,  1982). 

A  more  reasonable  hypothesis  is  that  larval  release  near  the  time  of  high  tide 
functions  as  adaptation  to  avoid  stressful  or  even  lethal  salinity  conditions.  Estuarine 
benthic  crabs  are  exposed  to  changes  in  salinity  over  a  tidal  cycle,  with  the  upper 
value  rarely  exceeding  35  ppt.  Salinity  tolerances  of  larvae  from  estuarine  crabs  are 
usually  sufficient  to  cope  with  the  maximum  salinity  values  they  are  likely  to  en- 
counter. For  example,  successful  larval  development  of  R.  harrisii  occurs  between 
2.5  and  40  ppt  (Costlow  et  al.,  1966).  The  real  tolerance  problem  is  low  salinity 
water,  which  would  be  experienced  at  low  tide.  Since  salinity  is  potentially  highest 
and  thereby  least  stressful  around  high  tide,  this  would  be  an  appropriate  time  for 
estuarine  crabs  to  release  their  larvae. 

Both  von  Hagen  (1970)  and  Saigusa  (1981)  also  consider  the  timing  of  larval 
release  to  be  related  to  salinity  tolerance.  They  both  suggest  that  if  larvae  are  released 
around  the  time  of  spring  high  tide,  the  subsequent  ebb  would  transport  them 
towards  the  ocean  where  they  would  encounter  less  stressful,  high  salinity  water. 
Since  R.  harrisii  larvae  are  not  transported  to  the  ocean,  larval  survival  may  depend 
upon  exposure  to  the  highest  possible  salinity  at  the  time  of  release. 

ACKNOWLEDGMENTS 

This  material  is  based  on  research  supported  by  the  National  Science  Foundation 
under  Grant  No.  OCE-77-26838.  We  thank  L.  Barrett  and  N.  J.  O'Connor  for 
assisting  with  the  preliminary  experiments,  and  Drs.  J.  Christy  and  M.  Salmon  for 
comments  on  the  manuscript.  We  also  appreciate  the  assistance  of  J.  Goy  and  S. 
Morgan  with  the  laboratory  habitat. 

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Rhythms  in  the  Marine  Environment,  P.  J.  DeCoursey,  Ed.  University  of  South  Carolina  Press, 

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Reference:  Biol.  Bull.  163:  301-319.  (October,  1982) 


GROWTH  AND  REGENERATION  PATTERNS  IN  THE  FIDDLER  CRAB, 

UCA  PUGILATOR 

PENNY  M.  HOPKINS 

Department  of  Zoology,  730  Van  Vleet  Oval,  University  of  Oklahoma,  Norman,  OK  73019 

ABSTRACT 

The  fiddler  crab,  Uca  pugilator,  will  survive  several  intermolt  cycles  in  the  lab- 
oratory, but  the  cycles  are  irregular.  Variations  in  cycles  are  due  to  variations  in  the 
length  of  stage  C4.  The  transition  from  C4  to  D  in  intact  crabs  does  not  seem  to  be 
due  to  environmental  clues  because  crabs  kept  in  constant  conditions  for  long  pe- 
riods of  time  continue  to  have  extremely  variable  intermolt  cycles. 

Multiple  autotomy  triggers  the  onset  of  proecdysis  and  a  post-autotomy  inter- 
molt cycle  that  is  significantly  shorter  than  controls.  Multiple  autotomy-induced 
proecdysis  is  divided  into  two  phases:  the  "reset  event"  is  independent  of  the  eye- 
stalks,  while  the  "proecdysial  program"  is  normally  under  their  control.  Loss  of 
a  cheliped  is  more  effective  in  initiating  a  reset  event  than  is  loss  of  a  single  walk- 
ing leg. 

Eyestalk  removal  forces  crabs  into  proecdysis.  If  crabs  are  in  early  proecdysis 
(stage  DO)  at  eyestalk  removal,  the  proecdysial  period  is  accelerated.  Eyestalk  removal 
results  in  large  increases  in  size  at  ecdysis  which  can  be  blocked  by  multiple  autot- 
omy. Ecdysis  does  not  always  result  in  growth.  Molting  in  Uca  may  result  only  in 
regeneration  of  missing  limbs.  Crabs  regenerating  a  number  of  limbs  may  actually 
become  smaller  at  molt. 

INTRODUCTION 

Ecdysis  of  the  calcified  exoskeleton  is  the  end  point  of  a  combination  of  phys- 
iological processes  used  by  decapod  crustaceans  to  achieve  both  general  body  growth 
and  regeneration  of  appendages.  Implicit  in  this  statement  is  the  assumption  that 
the  controls  of  ecdysis,  growth,  and  regeneration  are  intimately  linked  and  finely 
coordinated.  Ecdysis  and  regeneration  can  be  induced  during  non-growth  periods 
by  removal  of  the  eyestalks  or  of  many  appendages.  The  former  method  induces 
ecdysis  through  removal  of  inhibitory  neurosecretory  centers  in  the  eyestalk  (the  x- 
organ  and  sinus  gland).  The  removal  of  the  inhibitory  centers  usually  causes  a 
premature  ecdysis.  The  second  type  of  molt  induction  (called  multiple  autotomy) 
is  more  complicated  and  is  thought  to  involve  a  "resetting"  of  the  physiological 
processes  that  culminate  in  regeneration  and  ecdysis  (Skinner  and  Graham,  1972). 

The  fiddler  crab,  Uca  pugilator,  is  a  durable  and  exceptional  laboratory  animal. 
One  of  the  most  remarkable  features  of  these  hardy  little  crabs  is  the  single,  large 
cheliped  of  the  male  (from  which  this  entire  group  gets  its  common  name).  This 
cheliped  (or  claw)  is  often  longer  than  the  entire  carapace  of  the  crab.  One  third  of 
the  wet  weight  of  a  male  crab  may  be  due  to  the  cheliped.  The  cheliped  is  very 
important  in  social  and  reproductive  behavior  of  these  crabs  (Crane,  1975). 

Received  1  December  1981;  accepted  19  July  1982. 

Abbreviations:  C4,  intermolt  period  of  molt  cycle;  D,  proecdysial  period  of  molt  cycle;  E,  ecdysis; 
ER,  experimental  growth  rate;  MA,  multiple  autotomy  (including  cheliped);  MA-CI,  multiple  autotomy 
(cheliped  intact);  R3,  right  third  walking  leg;  R-value,  regeneration  index  value  for  R3. 

301 


302  PENNY  M.  HOPKINS 

The  fiddler  crab  has  been  used  by  many  investigators  in  various  physiological 
and  endocrinological  studies  (Abramowitz  and  Abramowitz,  1940;  Guyselman, 
1953;  Passano,  1960;  Vernberg  and  O'Hara,  1972;  Skinner  and  Graham,  1972; 
Fingerman  and  Fingerman,  1974;  Weis,  1976,  1977a,b).  Relatively  little  has  been 
reported,  however,  concerning  growth  and  molt  cycles  of  intact  animals  under  con- 
stant laboratory  conditions.  This  paper  describes  the  molting  cycles  in  intact  fiddler 
crabs  kept  in  constant  environmental  conditions  and  compares  these  "normal" 
cycles  to  autotomy-induced  and  eyestalk  removal-induced  cycles. 

This  paper  includes  observations  on:  (1)  the  effect  of  autotomy  of  various  num- 
bers of  limbs  upon  the  "reset"  and  duration  of  the  intermolt  cycle  and  growth 
patterns  of  the  carapace  and  limbs;  (2)  the  influence  of  autotomy  of  the  large  cheliped 
upon  the  intermolt  cycle;  (3)  the  effects  of  autotomy  and  eyestalk  removal  upon 
intermolt  cycles  subsequent  to  the  induced  cycles.  The  seemingly  contradictory 
effects  of  multiple  autotomy  upon  eyed  and  eyestalkless  crabs  has  been  investigated 
and  a  modified  model  for  autotomy-induced  proecdysis  is  proposed. 

MATERIALS  AND  METHODS 

Male  specimens  of  the  fiddler  crab,  Uca  pugilator,  were  obtained  from  the  Gulf 
Specimen  Company  of  Panacea,  Florida.  Shipments  were  received  throughout  the 
year.  Upon  arrival  in  the  laboratory,  the  crabs  were  forced  to  autotomize  the  right 
third  (R3)  walking  leg  (the  fourth  pereiopod)  by  pinching  the  merus  with  forceps. 
Individual  crabs  were  kept  in  transparent  plastic  boxes  (28  cm  X  17.5  cm  X  13.5 
cm)  with  a  small  amount  of  artificial  sea  water  (Instant  Ocean,  Aquarium  Systems, 
Inc.,  Menton,  Ohio).  Crabs  were  kept  in  environmental  chambers  maintained  at 
23°C  with  12  hours  of  illumination  each  day  beginning  at  6:00  AM.  The  crabs  were 
fed  oatmeal  once  a  week  and  allowed  to  feed  overnight.  The  water  in  the  boxes  was 
changed  the  following  day.  Animals  were  checked  daily  for  molts.  Crabs  were  allowed 
to  acclimate  in  the  laboratory  at  constant  environmental  conditions  for  at  least  two 
weeks  prior  to  being  used  in  any  experiment. 

The  carapace  width  of  each  animal  was  measured  with  a  vernier  caliper  (Mod- 
erntools,  MT-9).  The  regenerating  right  third  walking  leg  was  measured  every  other 
day  with  the  aid  of  an  ocular  micrometer  in  a  dissecting  microscope.  In  order  to 
compare  limbs  from  crabs  of  different  carapace  size,  the  length  of  a  regenerating 
limb  bud  was  converted  to  a  Regeneration  Index  (Bliss,  1956). 

length  of  limb  bud  (in  mm) 

Regeneration  Index  (R-value)  =  -  -  X  100 

carapace  width  (in  mm) 

Subdivisions  or  stages  of  intermolt  cycles  were  assigned  as  per  Skinner  (1962, 
after  Drach,  1939). 

The  length  of  the  large  cheliped  was  also  measured  with  the  vernier  caliper.  This 
measurement  is  the  linear  distance  from  the  notch  at  the  base  of  propodus  (at  the 
point  of  articulation  with  the  carpus)  to  the  tip  of  the  dactylus.  The  size  of  the 
cheliped  (in  mm)  was  divided  by  the  carapace  width  (in  mm)  and  this  number  is 
called  the  "Cheliped/Carapace  Ratio"  (C/C  Ratio). 

Following  the  emergence  of  the  right  third  limb  bud,  multiple  autotomy  of 
additional  walking  legs  and/or  the  large  cheliped  was  induced  as  described  above 
for  the  right  third  walking  leg. 

Eyestalks  were  removed  by  cutting  the  articulating  membrane  with  a  pair  of 
dissecting  scissors.  Prior  to  eyestalk  removal,  animals  were  anesthetized  by  cooling 
at  4°C  for  10  to  20  minutes. 


GROWTH  AND  REGENERATION  IN  CRABS  303 

Growth  rates  (ER's)  of  regenerating  limb  buds  were  calculated  as  previously 
described  (Bliss  and  Hopkins,  1974):  R3  values  are  plotted  against  time  (in  days). 
For  two  consecutive  R3  values,  the  slope  of  the  line  connecting  the  points  is  taken 
as  the  experimental  growth  rate  (ER)  for  the  limb.  The  slope  of  the  line  is  the  arc 
angle  of  the  sloped  line  relative  to  the  horizontal.  ER's  were  calculated  for  every 
day  of  an  intermolt  cycle.  The  average  ER  is  the  mean  of  those  daily  ER's. 

Water  content  of  chelipeds  and  walking  legs  was  determined  by  blotting  and 
weighing  the  limb  immediately  after  removal,  desiccating  in  a  drying  oven  for  four 
to  six  days,  then  weighing  again.  The  difference  in  weight  was  taken  as  the  water 
content  of  the  limb.  Protein  content  of  chelipeds  and  walking  legs  was  determined 
by  grinding  the  desiccated  limbs  in  ice  cold  5%  trichloroacetic  acid  (TCA)  in  a 
chilled  mortar  and  pestle.  The  solution  was  centrifuged  at  4°C  and  10,000  X  g  for 
20  minutes.  The  pellet  was  re-extracted  with  successive  extractions  in  80%  and  100% 
ethanol,  chloroform:ether  (2: 1  vol:vol),  and  ethyl  ether.  The  pellet  was  resuspended 
in  distilled  water  and  the  amount  of  protein  determined  by  the  method  of  Lowry 
et  al.  (1951)  using  bovine  serum  albumin  (Sigma  Chemical  Co.)  as  the  standard. 

Statistical  analysis  of  the  data  was  handled  as  follows:  means  were  determined 
and  the  homogeneity  of  variances  was  tested  using  the  Fmax  test  (Sokal  and  Rohlf, 
1969,  p.  370).  If  the  assumptions  for  normality  were  met,  analyses  were  done  using 
standard  analysis  of  variance.  However,  if  the  assumptions  of  analysis  of  variance 
were  not  met,  analogous  non-parametric  methods  (Mann- Whitney  U-test  and  Wil- 
coxon  two-sample  test)  were  used. 

RESULTS 
Intermolt  cycles  in  control  animals 

The  duration  of  intermolt  cycles  in  intact  crabs  varies  from  crab  to  crab  and 
from  cycle  to  cycle.  When  maintained  under  the  constant  laboratory  holding  con- 
ditions described  above,  Uca  pugilator  can  successfully  complete  as  many  as  six 
intermolt  cycles  (Table  I  and  Fig.  la).  The  durations  of  these  cycles  range  from  25 
to  1 7 1  days.  For  crabs  kept  in  the  lab  over  three  months,  the  range  is  34  to  1 36 
days.  Animals  maintained  under  constant  environmental  conditions  for  several 
months  show  some  reduction  in  the  mean  duration  of  the  intermolt  cycles.  The 
eventual  clustering  around  a  mean  intermolt  cycle  of  70  days  (Table  I)  is  the  result 
of  a  reduction  in  the  number  of  extremely  long  intermolt  cycles.  The  number  of 
shorter  cycles  is  unaffected. 

After  being  held  at  constant  conditions  for  several  months,  however,  individual 
crabs  continue  to  molt  independently  of  one  another:  there  are  no  "waves"  of 
molting.  The  pattern  of  variable  intermolt  durations  differs  from  one  crab  to  another. 
An  individual  crab  may  take  125  days  to  complete  one  intermolt  cycle  and  complete 
the  next  cycle  in  less  than  50  days.  Another  crab  in  identical  holding  conditions 
may  have  two  very  long  (or  very  short)  successive  intermolt  cycles  (Fig.  la).  The 
duration  of  a  single  intermolt  cycle  is  never  a  prediction  of  the  duration  of  subsequent 
cycles. 

In  Uca,  the  proecdysial  period  of  any  intermolt  cycle  requires  about  27  days 
(Table  II).  This  is  true  for  crabs  missing  only  one  limb  and  for  crabs  missing  eight 
limbs.  Crabs  that  are  destalked  during  C4  (see  Drach,  1939)  take  26.6  days  to  reach 
ecdysis.  Thus,  the  variations  observed  in  intermolt  cycle  lengths  represent  variations 
in  the  duration  of  stage  C4  rather  than  in  stage  D. 

These  differences  between  cycle  durations  of  crabs  that  have  been  in  the  lab 
under  identical  conditions  are,  in  part,  due  to  the  variations  in  the  sizes  of  the 


304 


PENNY  M.  HOPKINS 


TABLE  I 

Number  of  davs  (mean  ±  standard  error  of  the  mean)  from  initial  event  (either  autotomy  of  a  single  Rj 
walking  leg,  multiple  autotomy  or  eyestalk  removal)  to  the  first  ecdysis  in  the  lab. 


Controls 

(Lacking  a 
single  R3) 


Multiple  autotomy 


Eyestalkless 


Number  of 

days 
(±SEM) 


8  Walking  legs        Cheliped 
(MA-CI)  Intact 

Number  of 

days 
(±SEM)  n 


7  Walking  legs 
+  cheliped  (MA) 

Number  of 

days 
(±SEM)  n 


Number  of 

days 
(±SEM)  n 


Initial  event 

to 
Ecdysis  1 

Ecdysis  1 

to 
Ecdysis  2 

Ecdysis  2 

to 
Ecdysis  3 

Ecdysis  3 

to 
Ecdysis  4 

Ecdysis  4 

to 
Ecdysis  5 

Ecdysis  5 

to 
Ecdysis  6 


98.2  (±5.2)         75         26.0  (±1.1) 


85.0  (±4.2)         53         58.4  (±8.0) 


69.4  (±5.0)         27         79.3  (±19.0) 


23 


11 


70.2  (±7.3) 


68.8  (±7.8) 


76.4  (±5.8) 


17 


10 


10 


92.2  (±15.2) 


93.2  (±14.5) 


87.7  (±7.4) 


32.4  (±0.9)  89         22.7  (±0.8)         145 


67.0  (±3.9)  54         27.4  (±1.2)  28 


64.5  (±6.4)  19         28.0  (±1.4)  2 


67.8  (±7.9)  9 


40.5  (±6.9)  5 


99.3  (±40.3)  4 


The  mean  number  of  days  (±SEM)  for  subsequent  ecdyses  is  also  given.  The  number  of  crabs  in  each  group  is 
given  as  "n." 

animals.  When  the  duration  of  three  subsequent  intermolt  cycles  is  plotted  against 
the  initial  carapace  width  of  the  animal,  a  correlation  of  0.43  is  seen.  (This  correlation 
is  significant  at  P  <  0.01.)  For  example,  a  specific  animal  of  carapace  width  16.85 
mm  took  532  days  to  complete  three  intermolt  cycles  of  varying  durations.  Whereas, 
a  smaller  crab,  carapace  width  14.70  mm,  took  only  220  days  to  complete  three 
cycles.  The  pattern  of  alternating  short  and  long  intermolt  cycles,  however,  remains 
the  same  in  large  and  in  small  crabs. 


Intermolt  cycles  following  autotomy 

Autotomy  of  a  single  walking  leg  does  not  markedly  affect  the  duration  of  the 
intermolt  cycle  (Fig.  la).  Therefore,  crabs  missing  only  one  limb  are  referred  to  as 
"normal"  or  "controls"  throughout  this  report. 

The  duration  (and  variance)  from  autotomy  to  ecdysis  decreases  as  the  regen- 
eration load  is  increased  (Fig.  2  and  Table  III).  It  continues  to  decrease  until  the 
load  reaches  7  to  8  mg  of  protein.  The  regeneration  load  for  an  animal  is  calculated 
from  the  total  amount  of  protein  extracted  from  newly  regenerated  limbs  following 
ecdysis. 


GROWTH  AND  REGENERATION  IN  CRABS  305 

Multiple  autotomy  during  intermolt  cycle  stage  C4  significantly  hastens  the  next 
ecdysis  (Fig.  1  b.  Tables  I  and  II).  If  eight  walking  legs  are  autotomized  simultaneously 
and  the  cheliped  left  intact  (MA-CI),  the  length  of  time  from  autotomy  (=  initial 
event)  to  the  induced  ecdysis  is  significantly  shortened  when  compared  to  controls 
(Table  I).  However,  the  addition  of  the  large,  muscular  claw  to  the  regeneration  load 
(MA)  results  in  a  period  that  is  significantly  longer  (P  <  0.001)  than  the  comparable 
period  in  crabs  missing  only  eight  walking  legs  (Tables  I  and  II). 

The  influence  of  an  autotomized  cheliped  upon  the  induction  of  the  proecdysial 
period  (intermolt  stage  D)  seems  to  be  quantitatively  different  from  the  influence 
of  the  autotomy  of  a  single  walking  leg.  Autotomy  of  four  walking  legs  shortens  the 
time  from  autotomy  to  ecdysis  when  compared  to  controls  (Table  III).  However, 
autotomy  of  three  walking  legs  and  the  cheliped  results  in  a  significantly  faster  onset 
of  ecdysis.  Multiple  autotomy  of  seven  walking  legs  plus  the  cheliped  (MA)  is  more 
effective  in  prolonging  the  late  proecdysial  period  (D,)  than  multiple  autotomy  of 
eight  legs  only.  MA  prolonged  late  proecdysis  (stage  D!)  in  1 1  out  of  13  animals, 
while  MA-CI  was  effective  in  prolonging  D,  in  only  three  out  of  eight  animals 
(Table  II). 

Multiple  autotomy  also  has  a  pronounced  effect  on  the  second  post-autotomy 
intermolt  cycle  (Fig.  Ib  and  Table  I).  Not  only  is  the  immediately  induced  cycle 
affected  by  MA  and  MA-CI  but  also  the  second  post-autotomy  intermolt  cycle  is 
significantly  shorter  than  that  of  the  controls  (Table  I). 

Intermolt  cycles  following  eyestalk  removal 

Removal  of  eyestalks  hastens  ecdysis  (Tables  I  and  II).  When  eyestalks  are  re- 
moved from  crabs  that  have  spontaneously  entered  proecdysis  (stage  D0),  the  proec- 
dysial period  is  shortened  from  27.1  days  to  18.7  days  (Table  II).  Eyestalk  removal 
from  crabs  in  late  proecdysis  (stage  D,)  reduces  that  period  from  1 1.7  days  to  4.8 
days.  Thus,  it  appears  that  the  eyestalks  continue  to  exert  some  inhibitory  control 
during  most  of  the  proecdysial  period.  About  20%  of  eyestalkless  Uca  will  live 
through  a  second  molt  cycle.  These  crabs  molt  within  28  days  of  the  first  ecdysis 
(Table  I). 

Multiple  autotomy  of  seven  legs  and  cheliped  (MA)  after  eyestalk  removal  sig- 
nificantly prolongs  the  time  from  eyestalk  ablation  to  ecdysis  (Table  IV).  However, 
the  number  of  days  from  MA  to  ecdysis  (E)  is  less  than  comparable  periods  induced 
by  MA  in  intact  animals  (Table  IV).  In  fact,  the  time  from  MA  to  e  ;Iysis  in  eyestalk- 
less crabs  is  very  close  to  (and  statistically  indistinguishable  from)  the  time  from 
eyestalk  removal  to  ecdysis  of  otherwise  untreated  crabs  (Table  IV).  Thus,  MA  in 
eyestalkless  crabs  may  reset  the  proecdysial  period  but  does  not  have  any  effect  on 
the  duration  of  the  proecdysial  period  that  follows. 

Multiple  autotomy  during  late  proecdysis  in  eyestalkless  crabs  does  not  reset 
and  actually  speeds  the  proecdysial  period.  These  crabs  molt  more  quickly  than  do 
eyestalkless  controls  and  they  do  not  regenerate  any  of  the  newly  autotomized  limbs 
(Table  IV). 

Growth  patterns  in  control  animals 

Regeneration  of  walking  legs.  The  averaged  growth  pattern  of  several  right  third 
walking  limb  buds  is  illustrated  in  Figure  3  (solid  circles  and  solid  line).  The  first 
event  in  limb  regeneration  is  emergence  of  a  limb  bud  papilla  through  the  scar  tissue 
that  covers  the  coxal  stump.  The  time  between  autotomy  of  a  single  limb  and 


306 


PENNY  M.  HOPKINS 


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01     23456789 
Regeneration    Load 
(mg     of  protein) 


10 


FIGURE  2.  Intermolt  duration  (in  days)  as  a  function  of  regeneration  load  (=  sum  of  extractable 
protein  in  mg  from  all  regenerated  limbs  following  ecdysis).  Each  point  represents  the  mean  of  at  least 
ten  animals. 


emergence  of  the  papilla  is  quite  variable  in  control  crabs  (Table  III).  Limb  bud 
emergence  in  controls  takes  an  average  of  47%  of  the  total  cycle  regardless  of  the 
length  of  the  ensuing  intermolt  cycle. 

The  simultaneous  loss  of  two  or  four  walking  legs  hastens  the  emergence  of  all 
limb  buds  and  significantly  reduces  the  variances  of  emergence  time  (Table  III). 

TABLE  II 

The  effects  of  multiple  autotomy  of  eight  walking  legs  with  cheliped  left  intact  (MA-CI),  multiple 
autotomy  of  seven  walking  legs  plus  cheliped  (MA),  and  eyeslalk  removal  (ES)  at  intermolt  cycle  stages 
C4,  D0,  and  D,  on  the  mean  number  of  days  from  treatment  (T)  to  ecdysis  (E),  the  final  mean  Rj  R- 
value,  and  overall  growth  rate  (ER). 


Treat- 
ment 
(T) 

Molt  stage 
at  T  (after 
Skinner,  1962) 

Mean  R3 
value  at  T, 
(±SEM) 

Sample 
size  (n) 

Mean  number 
of  days  from 
T  to  ecdysis 
(E),  (±SEM) 

Mean  final 
R3  value 
(±SEM) 

Mean  overall 
growth  rate 
(ER)  T  to  E, 
(±SEM) 

Control 

C4 

0 

40 

96.8  (±5.4) 

22.50  (±0.5) 

18.1  (±1.9) 

MA-CI 

C4 

0 

14 

24.9  (±2.0) 

22.94  (±0.6) 

42.6  (±2.  3) 

MA 

C4 

0 

20 

29.7  (±2.0) 

21.48  (±0.4) 

35.5  (±3.7) 

SS 

c, 

0 

42 

26.6  (±1.3) 

21.35  (±0.9) 

39.0  (±1.6) 

Control 

Do 

13.11  (±0.2) 

42 

27.1  (±2.5) 

22.42  (±0.5) 

30.0  (±2.4) 

MA-CI 

Do 

12.80  (±0.5) 

15 

25.1  (±1.1) 

23.20  (±0.4) 

28.6  (±2.4) 

MA 

Do 

13.32  (±0.5) 

15 

28.0  (±1.5) 

23.00  (±0.4) 

28.0  (±2.4) 

ES 

Do 

12.52  (±0.4) 

24 

18.7  (±1.5) 

22.18  (±0.4) 

30.9  (±1.7) 

Control 

D, 

20.30  (±0.7) 

27 

11.7  (±2.0) 

24.00  (±0.4) 

20.9  (±5.6) 

MA-CI 

D, 

20.30  (±1.0) 

3 

2  1.0  (±0.6) 

22.48  (±1.1) 

9.8  (±1.7) 

D, 

23.16  (±1.3) 

5 

4.6  (±1.7)* 

23.  16  (±1.3) 

0 

MA 

D, 

2  1.48  (±3.2) 

11 

21.6  (±1.8) 

24.58  (±1.0) 

8.5  (±2.0) 

D, 

22.08  (±2.2) 

2 

5.5  (±1.5)* 

22.08  (±2.2) 

0 

BS 

D, 

20.82  (±1.0) 

6 

4.8  (±1.3) 

22.60  (±1.2) 

12.5  (±3.0) 

Means  are  given  ±  the  standard  error  of  the  mean. 
*  MA-CI  or  MA  limbs  not  regenerated. 


GROWTH  AND  REGENERATION  IN  CRABS 


309 


TABLE  III 

Autotomy-induced  reductions  in  means  (±  standard  error  of  means)  of  limb  bud  emergence  time 
and/or  reduction  in  the  variance  (V  =  (y  —  y)2/n  —  1)  of  bud  emergence  and  intermolt  cycle  durations. 


Mean  number  of  days  (±SEM) 


Variances 


Number  of  missing 

Autotomy 

Bud 

Autotomy 

limbs  (autotomized 

Sample 

to  bud 

emergence 

Autotomy 

to  bud 

Emergence 

Autotomy 

during  stage  C4) 

size  (n) 

emergence 

to  ecdysis 

to  ecdysis 

emergence 

to  ecdysis 

to  ecdysis 

1 

41 

43.5  (±3.6) 

65.8  (±5.0) 

110.4  (±5.8) 

518.7 

760.3 

1008.0 

2 

19 

18.0  (±2.0) 

48.5  (±8.9) 

72.8  (±6.7) 

70.3** 

719.8t 

491.1 

4 

1  1 

10.5  (±0.7) 

46.0  (±8.7) 

73.5  (±6.8) 

4.2! 

682.0tt 

510.8 

3  +  Cheliped 

16 

7.8  (±0.8) 

40.6  (±5.0) 

51.0  (±3.9) 

10.3 

327.8 

203.  1 

8  (MA-CI) 

37 

7.7  (±0.4) 

19.2  (±2.9) 

28.6  (±1.6) 

5.1 

75.8 

38.2 

7  +  Cheliped  (MA) 

35 

8.7  (±0.5) 

23.0  (±2.4) 

33.0  (±2.  3) 

8.4 

73.8 

91.7 

1* 

12 

8.7  (±0.7) 

57.1  (±8.7) 

60.1  (±8.6) 

5.4 

601.9 

598.8 

The  pooled  variance  ratios  were  calculated  to  test  the  equality  of  variance  and  the  variance  ratio  (F)  was  considered 
significant  at  P  <  0.05. 

Abbreviations  are  as  in  Table  II. 

*  Autotomized  following  MA-induced  ecdysis. 

**  F  =  7.4  (P  <  0.01). 

!F  =  16.7  (P  <  0.01). 

tF  =  1.06  (P>  0.05). 

ttF  =  1.06(P>  0.05). 

Following  the  emergence  of  the  limb  bud  papilla,  a  small  limb  bud  begins  to 
grow.  This  portion  of  limb  regeneration  is  called  basal  growth  (Bliss,  1956).  In  Uca, 
an  R3  bud  will  reach  R- values  of  10  to  13  during  basal  growth.  Basal  growth  in 
control  crabs  is  limited  to  stage  C4.  The  growth  rate  (ER)  of  the  limb  bud  during 
this  period  is  very  slow  (less  than  18)  and  the  small  amount  of  growth  that  does 
occur  may  occur  in  discontinuous  spurts. 

In  control  crabs,  rapid  proecdysial  growth  begins  at  approximately  75%  of  the 
entire  intermolt  cycle.  The  ER  of  the  limb  bud  may  reach  values  of  30  to  40  (Table 
II).  The  limb  bud  grows  and  differentiates,  and  the  muscles,  chromatophores,  and 


TABLE  IV 

The  effects  of  multiple  aulotomy  (seven  walking  legs  plus  cheliped  =  MA)  and  eyestalk  removal  (ES) 
performed  separately  (ES  or  MA)  or  together  (E8  plus  MA)  on  mean  intermolt  cycle  duration  (in 
days). 


Mean  initial  R3  value  (±SEM) 


Mean  number  of  days  (±SEM) 


at  ES 


at  MA 


Sample 
size  (n) 


ESto  E 


MA  to  E 


ES  plus 

0 

_ 

16 

27.6  (±2.2) 



MA 

0 

3.28  (±0.7) 

8 

33.3  (±1.5) 

24.0  (±1.3) 

0 

12.36  (±0.4) 

12 

36.2  (±1.4) 

20.1  (±0.6) 

0 

19.75  (±0.7) 

10 

22.0  (±1.8) 

6.1  (±1.2)* 

ESor 

0 

0 

42  20 

26.6  (±1.3) 

29.7  (±2.0) 

MA 

2.49  (±0.2) 

2.56  (±0.5) 

14  16 

33.4  (±1.9) 

29.4  (±1.7) 

11.  54  (±0.2) 

12.59  (±0.6) 

12  11 

20.2  (±1.9) 

27.6  (±1.7) 

20.82  (±1.0) 

20.23  (±0.7) 

4   11 

7.5  (±1.6) 

19.7  (±1.8) 

No  regeneration  of  MA  limbs. 


310 


PENNY  M.  HOPKINS 


10 


20 


1 


30          40          50  60  70 

%  of    Total  Intermolt  Cycle 


90 


100 


FIGURE  3.  Comparison  of  the  patterns  of  R,  limb  regeneration  in  controls  (solid  circles,  solid  line), 
animals  missing  seven  walking  legs  plus  cheliped  (  =  MA,  open  squares,  solid  line)  and  eyestalkless  crabs 
(crosses,  dashed  line).  The  arrow  represents  the  time  of  MA  and  eyestalk  ablation.  The  crossed  arrow 
indicates  the  point  at  which  eyestalkless  crabs  were  forced  to  autotomize  seven  walking  legs  plus  cheliped. 
The  subsequent  growth  pattern  is  shown  (open  circles,  dashed  line).  Each  point  represents  the  mean  of 
at  least  six  crabs,  and  the  vertical  lines  represent  standard  errors  of  the  means. 


segmentation  of  the  new  limb  become  visible  within  the  thin  cuticle  sac  that  covers 
the  bud. 

Regardless  of  the  length  of  the  intermolt  cycle,  the  final  R-values  of  the  limb 
buds  of  control  crabs  are  consistent  (Table  II).  A  long  cycle  does  not  result  in  a 
bigger  limb  bud  nor  does  confinement  to  a  short  cycle  limit  the  final  size  of  the 
bud.  A  limb  bud  continues  to  grow  until  ecdysis.  There  is  some  "terminal  plateau" 
(Bliss,  1956)  during  late  proecdysis  in  control  crabs  (Fig.  3).  The  ER's  of  the  limb 
bud  are  low  prior  to  ecdysis  (Table  II). 

At  an  R-value  of  22  to  23,  the  control  crabs  shed  the  old  exoskeleton.  As  the 
exoskeleton  is  discarded,  the  regenerated  bud  unfolds  and  expands.  The  only  visible 
differences  in  a  newly  regenerated  post-molt  walking  leg  are  its  slightly  smaller  size 
and  lighter  color. 

Carapace  and  cheliped  growth.  Following  molt,  the  new  carapace  of  control 
crabs  increases  in  width  by  2.4%  (Table  V).  The  average  increase  in  the  size  of  the 
cheliped  of  control  crabs  is  1.2%.  The  cheliped  increases  less  than  does  carapace 
width.  Therefore,  there  is  a  slight  reduction  of  the  cheliped/carapace  ratio  at  each 
ecdysis  in  control  crabs  (Table  VI).  A  cheliped  from  a  control  crab  contains  about 
50%  water  and  7.6%  protein  (Table  VII). 


Growth  patterns  following  autotomy 

Regeneration  of  walking  legs.  Multiple  autotomy  not  only  affects  the  duration 
of  the  induced  proecdysial  period,  it  also  has  an  effect  upon  the  pattern  of  growth 
of  the  regenerating  limb  buds  (Fig.  3).  The  limb  buds  of  MA  and  MA-CI  animals 
emerge  sooner  after  autotomy  than  do  the  limb  buds  of  control  animals  (Table  III), 
and  all  of  the  MA  and  MA-CI  buds  emerge  simultaneously.  The  average  rate  of 
growth  (ER)  of  R3  limb  buds  from  MA  crabs  autotomized  during  stage  C4  is  sig- 


GROWTH  AND  REGENERATION  IN  CRABS 


311 


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312 


PENNY  M.  HOPKINS 


TABLE  VI 

Changes  in  cheliped/carapace  ratios  (C/C)  following  three  successive  ecdyses. 

Mean  cheliped/carapace  ratios  (±SEM) 
Following  ecdyses  no: 


Limbs  missing: 

C/C  ratio 

Sample 
size  (n) 

C/C  ratio 

Saple 
size  (n) 

C/C  ratio 

Sample 
size  (n) 

1  Walking  leg 
8  Walking  legs 

(MA-CI) 
1  Cheliped 
3  Legs  +  cheliped 
7  Legs  +  cheliped 

(MA) 


1.43  (±0.02) 

1.41  (±0.04) 
0.69  (±0.02) 
0.71  (±0.02) 

0.69  (±0.01) 


43 

14 

1  \ 

12 

40 


1.35  (±0.03) 

1.36  (±0.03) 
0.83  (±0.02) 
0.90  (±0.04) 

0.82  (±0.02) 


23 

3 
9 
6 

27 


1.31  (±0.04) 

1.34  (±0.07) 
1.04  (±0.05) 
1.10  (±0.02) 

0.97  (±0.04) 


3 
6 
6 

10 


nificantly  higher  than  the  ER  of  R3  limb  buds  from  control  crabs  (Table  II).  However, 
if  MA  occurs  during  the  early  proecdysial  period  (D0),  the  ER  of  the  bud  is  no 
different  from  that  of  the  controls  (Table  II). 

The  final  R-values  of  R3  limb  buds  from  MA  crabs  are  the  same  as  the  final  R3 
values  for  the  controls  (Fig.  3  and  Table  II).  Yet,  the  post-ecdysial  size  of  newly 
regenerated  limbs  is  considerably  smaller  than  the  size  of  control  limbs  (Table  VII). 
A  non-regenerated  walking  leg  has  an  average  of  2.6  mg  of  protein,  and  a  regenerated 
R3  has  1.0  mg  of  protein.  However,  the  ratio  of  the  total  amount  of  protein/volume 
(=  propus  length3)  is  the  same  in  newly  regenerated  walking  legs  as  in  non-regen- 
erated legs  (Table  VII). 

Carapace  growth.  Following  a  multiple  autotomy-induced  ecdysis,  the  amount 
of  growth  (expressed  as  increase  in  carapace  width)  is  significantly  reduced  when 
compared  to  controls  (Table  V). 

Crabs  that  regenerate  eight  walking  legs  (MA-CI)  increase  only  0. 1 2%  in  carapace 
width.  Crabs  that  have  a  heavier  regeneration  load  (i.e.  seven  walking  legs  plus  the 
cheliped  =  MA)  actually  decrease  in  width  by  2.0%.  These  crabs,  however,  increase 
in  size  following  the  second  post-autotomy  ecdysis  and  continue  to  get  larger  at  each 
succeeding  ecdysis.  By  the  end  of  the  sixth  post-autotomy  cycle,  MA  crabs  are  not 
significantly  smaller  than  control  crabs  (Figs,  la  and  Ib). 

Regeneration  of  the  large  cheliped  can,  by  itself,  reduce  the  amount  of  post- 
autotomy  growth:  crabs  regenerating  a  single  walking  leg  and  a  cheliped  show  less 
increase  in  carapace  width  following  ecdysis  than  do  controls  (Table  V). 

Cheliped  growth.  A  regenerated  cheliped  is  always  very  small.  The  cheliped/ 
carapace  ratio  of  newly  regenerated  chelipeds  is  about  0.70  (Table  VI).  Crabs  are 
unable  to  regenerate  a  full-sized  cheliped  regardless  of  the  size  of  the  total  regen- 
eration load  (Table  V).  A  small  cheliped,  however,  grows  at  each  ecdysis  (Table  VI) 
and  the  ratio  of  protein  to  volume  is  not  significantly  less  than  the  ratio  for  non- 
regenerated  chelipeds  (Table  VII).  Crabs  that  have  lost  eight  walking  legs  but  not 
the  cheliped  are  able  to  maintain  the  growth  of  the  cheliped  at  each  ecdysis  (Tables 
V  and  VI).  These  crabs  do  not  appear  to  regenerate  walking  legs  at  the  expense  of 
the  cheliped.  The  percent  size  increase  of  the  cheliped  of  these  crabs  is  the  same  as 
the  percent  increases  of  controls.  The  regeneration  of  eight  walking  legs  is  accom- 
plished at  the  expense  of  carapace  growth  and  not  cheliped  growth  (Table  V). 


GROWTH  AND  REGENERATION  IN  CRABS 


313 


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3  1 4  PENNY  M.  HOPKINS 

A  large,  non-regenerated  cheliped  (C/C  ratio  =  1.66)  contains  an  average  of  32.2 
mg  of  extractable  protein  (Table  VII),  whereas  a  regenerated  cheliped  (average 
C/C  ratio  =  0.81)  has  an  average  of  1.8  mg  of  protein.  When  a  crab  loses  approx- 
imately 50  mg  of  protein  (32.2  mg  of  cheliped  protein  and  18-20  mg  of  walking 
leg  protein)  through  multiple  autotomy,  it  regenerates  only  8-9  mg  of  protein  (ap- 
proximately 1.8  mg  of  cheliped  protein  and  7-8  mg  of  walking  leg  protein). 

Growth  patterns  following  eyestalk  removal 

Regeneration  of  walking  legs.  Figure  3  illustrates  an  averaged  growth  curve  for 
regenerating  R3  limb  buds  from  eyestalkless  crabs  (crosses,  dashed  line).  Rapid  proec- 
dysial  limb  bud  growth  begins  soon  after  eyestalk  removal.  The  growth  curve  for 
this  R3  is  parallel  to,  but  ahead  of,  the  curve  for  control  crabs.  The  R3  limb  bud  of 
an  eyestalkless  crab  (like  the  limb  bud  of  a  MA  crab)  has  an  exaggerated  period  of 
no  growth  or  terminal  plateau  at  the  end  of  the  proecdysial  period  prior  to  ecdysis. 

Growth  of  an  R3  from  an  eyestalkless  crab  can  be  inhibited  during  C4  or  D0  by 
multiple  autotomy  (Fig.  3,  crossed  arrow).  The  inhibition  lasts  until  the  newly  au- 
totomized  papillae  emerge,  then  growth  of  all  limb  buds  continues  at  ER's  com- 
parable to  other  eyestalkless  crabs.  These  crabs  enter  terminal  plateau  at  R3  values 
significantly  lower  than  the  final  R3  values  of  intact  controls  and  eyestalkless  (but 
otherwise  untreated)  crabs  (Fig.  3). 

Frequently,  when  eyestalks  are  removed  at  the  same  time  as  autotomy,  the  limb 
bud  papilla  will  not  emerge  and  the  crab  will  molt  without  any  regeneration.  In 
most  of  the  experiments  reported  here,  the  R3  limb  papillae  were  allowed  to  emerge 
prior  to  eyestalk  removal.  In  about  25%  of  the  experimental  crabs,  eyestalk  removal 
did  not  cause  limb  bud  growth  or  ecdysis.  These  unresponsive  crabs  remained  alive 
for  considerable  lengths  of  time,  then  died.  They  generally  died  prior  to  the  ecdysis 
of  the  other  eyestalkless  crabs. 

Carapace  growth.  Eyestalk  removal  results  in  an  11.1%  increase  in  carapace 
width  (Table  V).  This  increase  is  reduced  to  7.4%  if  eyestalk  removal  is  followed 
by  multiple  autotomy  of  seven  walking  legs  plus  the  cheliped  (Table  V). 

Cheliped  growth.  Chelipeds  from  recently  molted,  eyestalkless  crabs  have  the 
same  linear  dimensions  as  do  the  chelipeds  from  control  crabs  (Table  VII).  However, 
the  chelipeds  from  eyestalkless  crabs  contain  relatively  less  protein  and  more  water 
than  do  the  chelipeds  from  controls,  and  the  ratio  of  the  amount  of  protein  to 
cheliped  volume  is  significantly  less  than  controls  (Table  VII).  When  the  cheliped 
and  several  walking  legs  are  autotomized  from  an  eyestalkless  crab,  the  regenerated 
cheliped  is  even  smaller  (C/C  =  0.34)  and  contains  much  less  protein.  The  protein 
to  volume  ratios  in  these  claws,  however,  are  similar  to  the  controls  (Table  VII). 

DISCUSSION 

When  male  specimens  of  Uca  pugilator  are  kept  in  the  laboratory  in  constant 
environmental  conditions  (23 °C,  12  hours  light/day,  private  boxes,  and  oatmeal 
once  per  week)  these  crabs  will  molt  and  grow.  The  intermolt  cycles  of  these  animals 
are  extremely  variable.  The  crabs  molt  independently  of  one  another  and  intermolt 
cycle  durations  vary  dramatically  from  crab  to  crab  and  from  cycle  to  cycle  (in 
intact  control  crabs  lacking  one  walking  leg).  If  Uca  are  held  in  the  lab  in  constant 
conditions  for  several  months,  there  is  a  reduction  in  the  mean  of  the  molt  cycle 
due  to  a  reduction  in  the  number  of  extremely  long  intermolt  periods.  The  mean 
of  these  later  intermolt  cycles  drops  to  about  70  days,  but  the  unpredictable  and 
variable  molting  patterns  for  individual  crabs  remain  unchanged. 


GROWTH  AND  REGENERATION  IN  CRABS  3  1  5 

The  crabs  used  in  these  experiments  were  collected  from  populations  of  crabs 
in  Florida.  The  climate  in  Florida  is  probably  less  of  a  limiting  factor  to  food  getting 
and  reproduction  than  in  more  temperate  regions.  Environmental  clues  serve  to 
synchronize  feeding,  reproductive,  and  molting  activities  of  some  populations.  Since 
natural  populations  of  Uca  molt  in  burrows  (away  from  other  members  of  the 
population)  and  females  copulate  in  a  hardened,  intermolt  stage  (rather  than  being 
restricted  to  the  shorter  and  softer  post-molt  stage)  there  would  be  no  obvious 
survival  or  reproductive  advantage  for  the  members  of  the  population  to  molt  in 
synchrony  (as  do  some  of  the  aquatic  crabs  and  shrimps).  It  is  not  surprising,  there- 
fore, that  external  clues  seem  to  be  less  important  in  controlling  intermolt  cycles 
in  Florida  populations  of  Uca  than  has  been  reported  for  other  crustaceans  (Bliss 
and  Boyer,  1 964;  Weis,  1 976).  Crane  ( 1 975)  has  suggested  that  much  of  the  ritualistic 
intermale  combat  and  courting  behavior  observed  in  populations  of  Uca  in  the  field, 
serves  to  synchronize  certain  group  activities.  The  vast  differences  in  intermolt  cycle 
durations  reported  here  may  be  due,  in  part,  to  the  fact  that  these  experimental 
crabs  were  held  in  individual  boxes.  Crabs  held  apart  are  deprived  of  any  social 
synchronization. 

Although  individual  crabs  held  in  constant  conditions  continue  to  molt  inde- 
pendently of  one  another,  they  can  be  induced  (by  multiple  autotomy  and  eyestalk 
removal)  to  enter  proecdysis  and  molt  in  concert.  However,  the  two  induced  proec- 
dyses  are  very  different:  while  MA  and  MA-CI  seem  to  reset  a  highly  controlled  and 
biphasic  program,  eyestalk  removal  appears  to  simply  remove  endogenous  inhibitory 
mechanisms  (that  in  control  animals  are  withheld  only  during  late  proecdysis). 

The  response  to  multiple  autotomy  in  Uca  is  divided  into  two  distinct  phases. 
The  first  phase  consists  of  a  physiological  resetting.  In  Uca,  the  "reset  event"  is  (1) 
independent  of  the  eyestalks;  (2)  inhibitory  to  proecdysis;  and  (3)  the  initial  response 
to  autotomy.  Skinner  and  Graham  (1972)  suggested  that  multiple  autotomy  in  crabs 
resets  the  entire  intermolt  cycle.  In  Uca,  this  does  not  seem  to  be  the  case.  It  appears 
that  the  reset  effect  of  multiple  autotomy  is  independent  of  the  effect  of  multiple 
autotomy  upon  the  duration  of  the  subsequent  proecdysial  period.  The  number  of 
days  from  MA  (or  MA-CI)  to  ecdysis  is  consistent  regardless  of  whether  MA  occurs 
during  C4  or  early  D  (Table  II).  But  when  MA  occurs  in  eyestalkless  animals,  only 
the  reset  effect  is  observed.  MA  seems  to  have  no  effect  on  the  proecdysial  program 
when  eyestalks  are  missing. 

In  Uca,  autotomy-induced  resetting  allows  for  the  emergence  and  early  growth 
of  autotomized  limb  buds.  Adiyodi  (1972)  has  shown  that  the  earliest  phase  of 
regeneration  (limb  bud  emergence  and  basal  growth)  in  the  crab  Pamtelphusa  is 
characterized  by  extensive  mitotic  activity  and  is  different  from  the  actual  proecdysial 
growth  phase  which  is  characterized  by  increased  cell  size  rather  than  number.  Limb 
bud  emergence  and  basal  growth  are  independent  of  proecdysis  and  are  inhibited 
if  autotomy  occurs  during  the  later  stages  of  D  (Bliss,  1956;  Passano  and  Jyssum, 
1963;  Hopkins,  et  al.,  1979).  Thus,  when  a  limb  is  lost,  it  is  necessary  to  establish 
the  internal  physiological  conditions  that  will  allow  for  the  mitotic  events  of  blastema 
organization  and  limb  bud  papillae  emergence.  If  the  function  of  the  reset  event  is 
to  allow  blastema  organization  and  early  bud  growth,  then  the  reset  event  is  not 
limited  to  multiple  autotomy.  The  loss  of  a  second  walking  leg  during  C4  has  a 
profound  effect  on  the  growth  of  the  previously  autotomized  limb  bud.  The  basal 
growth  of  the  first  limb  bud  is  inhibited  until  the  emergence  of  the  second  limb 
papilla.  Both  of  these  limb  buds  will  then  proceed  through  basal  growth  simulta- 
neously. The  duration  from  autotomy  until  emergence  of  the  second  papilla  is 
significantly  shorter  than  the  time  for  emergence  of  the  first  limb.  The  simultaneous 


3  1  6  PENNY  M.  HOPKINS 

loss  of  two  limbs  during  C4  hastens  the  emergence  of  both  limb  papillae  (Table  III). 
Autotomy  of  two  limbs  has  a  reset  effect  that  is  less  than  the  effect  of  autotomy  of 
four  limbs  or  of  MA. 

The  resetting  event  that  allows  for  emergence  of  the  blastema  also  seems  to  have 
an  effect  on  the  time  that  it  takes  the  animal  to  reach  proecdysis.  There  is  a  decrease 
in  the  number  of  days  from  autotomy  to  ecdysis  with  increasing  numbers  of  limbs 
removed.  Thus,  there  is  a  cumulative  effect  of  limb  loss  upon  the  onset  of  proecdysial 
program  in  Uca.  Each  limb  adds  to  the  overall  effect.  Fingerman  and  Fingerman 
(1974)  have  reported  in  female  Uca  pugilator,  an  increase  in  molting  rate  (expressed 
as  percent  ecdysis/time)  with  increased  numbers  of  limbs  removed.  Weis  (1977b) 
reported  that  multiple  autotomy  during  early  proecdysis  (R,  value  of  10)  accelerated 
the  growth  of  the  original  R,  and  hastened  the  onset  of  ecdysis  in  Uca.  She  also 
reported  that  autotomy  of  five  or  more  limbs  had  a  greater  acceleratory  effect  than 
autotomy  of  two  limbs.  In  describing  the  effects  of  limb  loss  on  molt  cycle  in  the 
cockroach,  Blattella,  Kunkel  (1977)  suggested  that  there  is  an  independent  signal 
from  each  regenerating  limb  with  an  average  delay  message  programmed  for  each 
autotomized  limb  in  the  hemiganglion  serving  that  limb.  A  similar  model  may  be 
applicable  to  Uca,  with  each  limb  having  an  individual  message  and  the  final  effect 
being  the  sum  of  those  messages. 

The  extremely  large  cheliped  of  Uca  has  a  greater  resetting  effect  than  does  a 
single  walking  leg.  Emergence  of  limb  papillae  in  response  to  autotomy  of  four 
walking  legs  lags  behind  limb  papillae  emergence  in  response  to  loss  of  three  walking 
legs  and  the  cheliped.  Also,  autotomy  of  eight  walking  legs  is  less  effective  in  causing 
a  reset  event  in  late  proecdysis  than  is  autotomy  of  seven  walking  legs  plus  the 
cheliped.  These  results  differ  from  those  reported  for  the  tropical  land  crab,  Gecar- 
cinus  lateralis  (Skinner  and  Graham,  1972).  In  Gecarcinus,  loss  of  a  cheliped  was 
no  more  effective  than  loss  of  a  walking  leg  in  inducing  proecdysis.  The  large  cheliped 
of  Uca,  however,  is  relatively  much  larger  than  either  of  the  chelipeds  of  Gecarcinus 
and  may  play  a  more  important  role  in  the  social  and  reproductive  behavior  of  Uca 
than  do  the  two  chelipeds  of  Gecarcinus.  Therefore,  there  may  be  a  greater  advantage 
to  Uca  to  preferentially  regenerate  the  cheliped. 

The  second  phase  of  an  autotomy-induced  cycle  is  the  actual  growth  phase  of 
"proecdysial  program."  This  program  is  (1)  normally  under  the  control  of  the  eye- 
stalks;  and  (2)  disrupted  by  the  reset  event.  The  proecdysial  duration  of  crabs  missing 
eight  walking  legs  (MA-CI)  is  the  same  as  that  of  crabs  missing  their  eyestalks  and 
of  control  crabs  (25  to  27  days).  This  is  a  significantly  shorter  duration  than  the 
duration  from  MA  to  ecdysis  in  eyed  crabs  (33  days).  If  25-27  days  represents  the 
shortest  proecdysial  duration,  then  loss  of  the  cheliped  must  exert  some  inhibitory 
control  over  the  onset  or  duration  of  the  proecdysial  program.  This  inhibitory  control 
is  mediated  through  the  eyestalks  because  MA  of  eyestalkless  crabs  resets  but  does 
not  affect  the  proecdysial  program.  Likewise,  MA  during  D,  in  intact  crabs  resets 
but  does  not  affect  the  subsequent  proecdysial  program.  Thus,  in  crabs  with  minimal 
(or  no)  eyestalk  controls,  MA  can  only  initiate  the  reset  event  and  has  no  control 
over  the  proecdysial  program. 

Eyestalk  removal  in  Uca  does  not  always  result  in  regeneration  and  ecdysis.  Up 
to  25%  of  destalked  Uca  do  not  respond  to  eyestalk  removal.  Charmantier-Daures 
(1976)  reported  that  during  stage  C4,  eyestalk  removal  in  the  crab,  Pachygrapsus, 
induced  regeneration  in  only  50%  of  the  crabs.  Perhaps,  these  unresponsive  crabs 
are  physiologically  inadequate  to  initiate  the  processes  that  lead  to  ecdysis.  Unlike 
the  crab  Gecarcinus,  eyestalkless  Uca  do  not  always  die  at  or  before  molt.  About 
20%  of  eyestalkless  Uca  live  through  two  ecdyses  and  the  length  of  the  second 
intermolt  is  virtually  the  same  as  the  first  intermolt  duration. 


GROWTH  AND  REGENERATION  IN  CRABS  3  1  7 

It  has  been  proposed  that  the  effects  which  follow  autotomy  in  crustaceans  are 
due  to  the  severance  of  a  critical  number  of  leg  nerves  (Skinner  and  Graham,  1972; 
Bittner  and  Kopanda,  1973).  This  "severed  nerve  hypothesis"  would  not,  however, 
account  for  the  fact  that  in  Uca  autotomy  of  the  cheliped  has  a  greater  effect  than 
autotomy  of  a  single  leg.  Nor  could  it  account  for  the  fact  that  the  duration  of  the 
second  post-autotomy  intermolt  cycle  is  significantly  shorter  than  the  comparable 
intermolt  cycle  of  the  controls.  (Charmantier-Daures,  1976,  observed  a  similar  effect 
in  the  crab,  Pachygrapsus.)  These  facts  suggest  that  a  message  with  qualitative  and 
quantitative  information  about  the  limb  is  conveyed  to  the  CNS  and  the  message 
is  not  merely  an  on/off  signal  as  suggested  by  the  severed  nerve  hypothesis.  Newly 
regenerated  limbs  are  smaller  after  molt  than  non-regenerated  limbs  (see  below)  and 
slight  injuries  may  occur  to  the  new  limbs  during  the  extremely  difficult  task  of 
getting  out  of  an  old  exoskeleton  with  a  minimum  number  of  limbs  and  efficiency. 
Minor  injuries  and/or  small  limb  size  may  alter  or  modify  the  messages  sent  back 
to  the  CNS  by  the  intact  limbs.  The  "program"  may  also  respond  to  sensory  input: 
smaller,  newly  regenerated  limbs  may  not  have  as  many  sensory  structures  as  non- 
regenerated  limbs. 

The  effects  of  MA  (and  MA-CI)  are  evident  in  the  growth  rates  of  the  regenerating 
limb  buds.  MA  during  intermolt  speeds  the  ER's  of  the  resulting  limb  buds.  During 
mid-proecdysis,  the  rates  of  growth  are  unaffected  and  in  late  proecdysis  the  overall 
rates  of  limb  bud  growth  are  slowed.  The  final  size  of  the  regenerated  limb  bud  does 
not  appear  to  be  affected  by  speeding  or  slowing  the  growth  rates.  The  final  size  of 
the  limb  buds  are  the  same  for  limb  buds  that  have  regenerated  slowly  and  buds 
that  have  regenerated  quickly. 

In  Uca,  ecdysis  does  not  always  result  in  an  increase  in  carapace  size  (see  also 
Guyselman,  1953;  Weis,  1976).  Ecdysis  may  take  place  solely  as  a  means  of  regen- 
erating missing  limbs,  and  sometimes  regeneration  may  take  place  at  the  expense 
of  general  body  growth.  Under  the  holding  conditions  described  here,  crabs  that 
regenerate  more  than  four  legs  possess  a  new  carapace  that  is  no  larger  and  some- 
times smaller  than  the  one  shed.  There  is  a  relationship  between  regeneration  load 
and  degree  of  growth  (or  no  growth)  observed  in  the  post-molt  carapace.  Fingerman 
and  Fingerman  (1974)  reported  that  intact  female  Uca  regenerating  eight  walking 
legs  showed  less  growth  than  intact  crabs  missing  only  one  limb,  but  they  did  not 
report  any  loss  of  carapace  size.  The  new  exoskeleton  of  a  post-ecdysial  crab  is 
initially  expanded  with  water  taken  up  and  stored  during  proecdysis  (Bliss  and  Boyer, 
1964)  and  during  post-molt  the  fluid  is  replaced  with  protein  (Skinner,  1966).  Per- 
haps the  volume  of  water  taken  up  during  proecdysis  is  the  same  whether  the  crab 
is  or  is  not  regenerating  limbs.  During  post-molt,  then,  an  MA  crab  must  use  that 
volume  of  water  to  expand  not  only  the  new  exoskeleton  carapace  but  also  the 
newly  regenerated  cheliped  and  all  of  the  new  walking  legs.  The  reduction  in  carapace 
size  (or  lack  of  increase  in  size)  might,  therefore,  be  due  to  insufficient  water  uptake 
during  proecdysis. 

The  failure  to  increase  in  size  at  ecdysis  is  not  due  to  the  truncated  proecdysis. 
Eyestalkless  crabs  have  the  briefest  proecdysial  duration,  yet  eyestalkless  crabs  have 
the  largest  post-ecdysial  increase  in  carapace  size.  MA  reduces  the  post-molt  increase 
in  size  of  eyestalkless  crabs.  If  the  increase  in  carapace  size  in  eyestalkless  animals 
is  due  to  increased  water  uptake,  then  MA  may  block  the  increase  in  carapace  size 
in  much  the  same  way  that  it  may  block  the  increase  in  intact  crabs. 

Intact  control  crabs  do  not  always  have  a  terminal  plateau  at  the  end  of  the 
proecdysial  period.  Terminal  plateau  (a  period  of  no  growth  preceding  ecdysis) 
occurs  consistently  in  eyestalkless  and,  to  a  lesser  extent,  in  MA  and  MA-CI  crabs. 
Crabs  missing  seven  or  eight  limbs  show  some  terminal  plateau,  but  less  terminal 


3  1  8  PENNY  M.  HOPKINS 

plateau  is  evident  in  crabs  missing  fewer  limbs.  Perhaps  regeneration  becomes  un- 
coupled from  other  proecdysial  events  in  those  crabs  that  have  an  exaggerated  ter- 
minal plateau.  The  fact  that  eyestalkless  crabs  (with  subsequent  MA)  have  a  terminal 
plateau  at  R-values  that  are  significantly  lower  than  in  eyestalkless  crabs  suggests 
that  terminal  plateau  is  not  due  to  limb  buds  having  reached  maximal  size,  but 
rather  is  due  to  physiological  conditions  at  the  end  of  proecdysis  that  are  inhibitory 
to  further  growth  of  the  limb  buds.  At  ecdysis,  eyestalkless  crabs  have  buds  that  are 
the  same  size  as  the  limb  buds  of  intact  crabs  at  ecdysis.  The  fact  that  these  buds 
are  no  smaller  than  other  buds  is  unexpected  in  light  of  the  extreme  differences 
found  in  size  and  protein  content  of  the  post-ecdysial  limbs. 

It  has  been  reported  in  other  crabs  that  post-molt  regenerated  limbs  are  smaller 
than  post-molt  non-regenerated  limbs  (Skinner  and  Graham,  1972;  Charmantier- 
Daures,  1976).  This  is  also  true  in  Uca.  Fingerman  and  Fingerman  (1974)  and  Weis 
(1976)  also  reported  that  post-molt  walking  legs  were  smaller  in  MA  Uca.  Newly 
regenerated  legs  are  32%  smaller  than  non-regenerated  legs  and  contain  62%  less 
protein.  In  Uca  a  regenerated  cheliped  is  much  smaller.  Newly  regenerated  chelipeds 
increase  in  size  with  each  succeeding  ecdysis.  The  chelipeds  increase  28%  at  the 
second  post-autotomy  ecdysis  and  continue  to  increase  at  each  ecdysis.  Due  to  the 
high  mortality  rate  for  MA  crabs,  it  was  never  observed  whether  the  regenerated 
chelipeds  ever  regain  their  former  dimensions. 

Under  the  holding  conditions  described  above,  Uca  is  capable  of  de  novo  syn- 
thesis of  only  9  mg  of  protein  (regardless  of  how  many  limbs  were  lost  through 
autotomy).  This  amount  of  protein  is  much  less  than  the  amount  the  crab  Gecarcinus 
is  capable  of  regenerating  (Skinner  and  Graham,  1972).  However,  this  difference 
may  be  due  to  the  fact  that  Gecarcinus  is  a  considerably  larger  crab. 

Skinner  (1966)  reported  that  the  amount  of  muscle  per  cheliped  in  Gecarcinus 
was  lowest  during  the  first  few  days  after  ecdysis  and  the  maximal  growth  of  the 
chelipeds  (in  terms  of  incorporation  of  14C-leucine  into  protein)  occurred  during 
post-molt.  The  post-molt  size  of  an  unregenerated  cheliped  from  an  eyestalkless  Uca 
has  the  same  linear  dimensions  as  the  unregenerated  cheliped  from  an  eyed  control 
crab.  However,  the  ratio  of  protein  to  volume  of  the  cheliped  from  the  eyestalkless 
crab  is  greatly  reduced.  These  chelipeds  from  eyestalkless  crabs  grow  over  5%  in 
linear  dimensions  following  ecdysis  but  contain  much  less  protein.  This  is  probably 
due  to  the  fact  that  these  eyestalkless  crabs  have  little  or  no  post-molt,  but  rather 
pass  very  quickly  from  ecdysis  into  a  new  proecdysial  period.  Thus,  eyestalkless 
crabs  have  less  "down  time"  in  which  muscle  protein  can  be  synthesized  to  replace 
muscle  protein  autolysed  during  proecdysis.  On  the  other  hand,  eyestalkless  crabs 
that  are  subsequently  autotomized  (including  the  cheliped)  are  sufficiently  inhibited 
by  the  resetting  action  of  autotomy  that  they  can  regenerate  the  cheliped.  The  period 
of  regeneration  is  so  short,  however,  that  the  linear  dimensions  of  the  newly  regen- 
erated cheliped  are  only  half  the  dimensions  of  chelipeds  regenerated  by  intact  crabs. 
Perhaps  the  physiological  conditions  of  proecdysis  are  inhibitory  to  protein  synthesis, 
or  the  autolysis  of  muscle  that  occurs  during  proecdysis  is  so  extensive  that  it  some- 
how overrides  most  synthetic  efforts. 

LITERATURE  CITED 

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Cytol.  32:  257- 
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CRANE,  J.  1975.  The  Fiddler  Crabs  of  the  World,  Ocypodidae:  Genus  Uca.  Princeton  University  Press, 

Princeton,  New  Jersey. 
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19:  103-391. 
FINGERMAN,  M.,  AND  S.  W.  FiNGERMAN.  1974.  The  effects  of  limb  removal  on  the  rates  of  ecdysis  of 

eyed  and  eyestalkless  crabs,  Uca  pugilator.  Zool.  jb.  Physiol.  78:  301-309. 
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HOPKINS,  P.  M.,  D.  E.  BLISS,  S.  W.  SHEEHAN,  AND  J.  R.  BOYER.  1979.  Limb  growth-controlling  factors 

in  the  crab  Gecarcinus  lateralis,  with  special  references  to  the  limb  growth-inhibiting  factor. 

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KUNKEL,  J.  G.  1977.  Cockroach  molting.  II.  The  nature  of  regeneration-induced  delay  of  molting  hor- 
mone secretion.  Biol.  Bull.  153:  145-162. 
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the  Folin  phenol  reagent.  /.  Biol.  Chem.  193:  266-275. 
PASSANO,  L.  M.  1960.  Molting  and  its  control.  Pages  473-576  in  The  Physiology  of  Crustacea,  T.  H. 

Waterman,  Ed.  Vol.  1 .  Academic  Press,  New  York  and  London. 

PASSANO,  L.  M.,  AND  S.  JYSSUM.  1963.  The  role  of  the  Y-organ  in  the  crab  proecdysis  and  limb  regen- 
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SKINNER,  D.  M.  1962.  The  structure  and  metabolism  of  a  crustacean  integumentary  tissue  during  a  molt 

cycle.  Biol.  Bull.  123:  635-647. 
SKINNER,  D.  M.  1966.  Breakdown  and  reformation  of  somatic  muscles  during  the  molt  cycle  of  the  land 

crab,  Gecarcinus  lateralis.  J.  Exp.  Zool.  163:  1 15-124. 
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Reference:  Biol.  Bull.  163:  320-328.  (October,  1982) 


FINE  STRUCTURE  OF  A  SCYPHOZOAN  PLANULA, 
CASSIOPEIA  XAMACHANA 

VICKI  J.  MARTIN1  AND  FU-SHIANG  CHIA2 

1  Department  of  Biology,  University  of  Louisville.  Louisville,  KY  40292,  and  2  Department  of  Zoology, 
University  of  Alberta,  Edmonton,  Alberta,  Canada  T6G  2E9 

ABSTRACT 

Pre-metamorphic  planulae  of  the  scyphozoan  Cassiopeia  xamachana  contain 
four  cell  types.  The  ectoderm  consists  of  supportive  cells  and  differentiating  ne- 
matoblasts  and  nematocytes,  while  the  endoderm  consists  of  supportive  cells  and 
interstitial  cells.  Neural  elements  and  glandular  cells  are  absent  in  these  planulae. 
Morphological  similarities  and  differences  that  exist  among  hydrozoan,  scyphozoan, 
and  anthozoan  planulae  are  discussed. 

INTRODUCTION 

Most  cnidarians  have  a  planula  stage  at  some  time  in  their  life  cycle.  Planulae 
are  cylindrical  and  are  composed  of  an  ectoderm  and  an  endoderm  separated  by 
a  thin  mesoglea.  In  recent  years  several  ultrastructural  studies  have  described  the 
morphology  of  hydrozoan  and  anthozoan  planulae  (Lyons,  1 973a,  b;  Vandermeulen, 
1974;  Chia  and  Crawford,  1977;  Martin  and  Thomas,  1977,  1980;  Chia  and  Koss, 
1979).  The  ultrastructural  morphology  of  scyphozoan  planulae  has  been  largely 
ignored.  Otto  (1978)  examined  the  morphological  and  ultrastructural  changes  which 
took  place  during  settlement  of  scyphozoan  planulae  of  Haliclystus  salpinx.  The 
planulae  of  this  Stauromedusae  are  atypical  in  that  they  lack  cilia,  do  not  swim, 
and  usually  contain  a  constant  number  of  endodermal  cells.  Since  there  has  been 
no  comprehensive  fine-structural  study  to  date  describing  a  more  typical  scyphozoan 
planula,  we  examined  the  planulae  of  Cassiopeia  xamachana.  It  is  hoped  that  such 
a  study  might  reveal  possible  morphological  similarities  and  differences  among  hy- 
drozoan, scyphozoan,  and  anthozoan  planulae. 

MATERIALS  AND  METHODS 

Adult  Cassiopeia  were  collected  in  December,  1980  at  La  Paguera,  Puerto  Rico. 
Gonads  and  gastric  filaments  were  removed  from  the  adults,  placed  in  finger  bowls 
of  filtered  sea  water,  and  macerated  with  a  pipette.  Young  planulae  were  soon 
observed  swimming  in  these  containers.  Four  days  after  collection  of  planulae,  swim- 
ming planulae  were  fixed  for  2'/2  hours  in  2.5%  glutaraldehyde  in  0.2  M  phosphate 
buffer  (Dunlap,  1966;  Cloney  and  Florey,  1968).  They  were  post-fixed  for  2  hours 
in  2%  osmium  tetroxide,  pH  7.2,  in  1.25%  sodium  bicarbonate  (Wood  and  Luft, 
1965).  Specimens  for  transmission  electron  microscopy  were  dehydrated  in  an 
ethanol  series,  infiltrated  with  propylene  oxide,  and  embedded  in  Epon  (Luft,  1961). 
Blocks  were  sectioned  on  a  Porter  Blum  MT-2B  ultramicrotome,  placed  on  1 50- 
mesh  copper  grids,  and  stained  in  5%  uranyl  acetate  in  methanol  followed  by  lead 
hydroxide.  Grids  were  examined  with  a  Phillips  EM  201  transmission  electron 

Received  26  February  1982;  accepted  13  July  1982. 

320 


CASSIOPEIA  FINE  STRUCTURE  321 

microscope.  Planulae  fixed  for  scanning  electron  microscopy  were  dehydrated 
through  a  graded  series  of  amyl  acetates,  critical  point  dried,  mounted  on  stubs  and 
shadowed  with  carbon  followed  by  gold.  The  specimens  were  examined  with  a 
Cambridge  Stereoscan  150  SEM. 

For  histochemical  studies  and  the  detection  of  glandular  cells,  thick  plastic  serial 
sections,  1-3  /um  thick,  were  mounted  on  glass  slides.  The  Epon  was  removed 
according  to  the  method  of  Lane  and  Europa  (1965)  and  the  sections  were  stained 
by  the  periodic  acid-Schiff  (PAS)  procedure  (Lillie,  1954). 

RESULTS 

The  pre-metamorphic  planula  of  Cassiopeia  ranges  from  120  ^m  to  220  nm  in 
length  and  from  85  nm  to  100  jum  in  width  in  its  mid-region.  It  is  uniformly  ciliated 
and  swims  with  the  enlarged  anterior  end  forward.  Just  prior  to  metamorphosis,  an 
indentation  is  found  at  the  anterior  end  (Fig.  1).  The  majority  of  planulae  observed 
settle  on  the  bottoms  of  glass  dishes  and  undergo  metamorphosis  within  4-5  days 
after  collection.  In  some  cases,  planulae  undergo  metamorphosis  without  prior  at- 
tachment to  glass. 

Fine-structural  examination  of  pre-metamorphic  planulae  reveals  only  4  cell 
types:  2  in  the  ectoderm  and  2  in  the  endoderm.  The  ectoderm  consists  of  supportive 
cells  and  differentiating  nematoblasts  and  nematocytes.  Supportive  cells  are  colum- 
nar in  shape  and  extend  from  the  free  surface  of  the  planula  to  the  mesoglea  (Fig. 
2).  Each  supportive  cell  bears  microvilli  and  a  single  cilium  at  its  apical  surface 
(Figs.  2  and  3).  The  cilium  is  of  the  9  +  2  microtubular  arrangement  and  extends 
from  the  apical  surface  without  a  concavity.  It  consists  of  a  basal  plate  located  above 
a  basal  body  and  an  accessory  basal  body  (Fig.  4).  The  basal  body  gives  rise  to  a 
striated  ciliary  rootlet  with  a  periodic  banding  pattern  of  about  300  A.  The  root- 
let extends  deep  into  the  cytoplasm  of  the  cell  and  terminates  just  above  the  nucleus. 
Attached  to  the  accessory  basal  body  is  a  plaque-like  structure  that  parallels  the 
ciliary  rootlet  (Fig.  4).  Microfilaments  of  a  terminal  web  are  found  directly  beneath 
the  apical  surfaces  of  the  cells  (Fig.  5)  and  terminate  at  the  lateral  cell  boundaries 
on  either  side.  Septate  desmosomes  are  present  between  these  supportive  cells  in 
their  apical  regions  (Fig.  6).  Numerous  electron-dense,  membrane-bounded  granules 
fill  the  apical  regions  of  the  cells  (Fig.  2).  Vacuoles  are  also  present.  The  nucleus  of 
each  cell  is  centrally  located  and  contains  a  nucleolus  and  condensed  chromatin. 
A  few  Golgi  complexes  are  located  in  close  association  with  the  nucleus.  Mito- 
chondria, polysomes,  and  endoplasmic  reticulum  are  scattered  throughout  the  cy- 
toplasm. 

Basally,  foot  processes  of  the  supportive  cells  insert  on  the  mesoglea  (Fig.  7). 
PAS-positive  granules  and  glycogen  particles  are  abundant  in  the  basal  regions  of 
these  cells.  Specialized  junctional  complexes  resembling  desmosomes  and  hemi- 
desmosomes  are  located  between  the  foot  processes  of  adjacent  supportive  cells  and 
between  the  foot  processes  and  the  mesoglea  (Fig.  8).  Microfilaments  are  seen  ra- 
diating out  from  dense  regions  located  along  the  inner  borders  of  the  junctional 
membranes  (Fig.  9).  The  two  membranes  are  separated  by  a  space  of  150-200  A. 

Fully  differentiated  nematocytes  are  abundant  at  the  ectodermal  surfaces  of 
planulae  (Fig.  10).  They  are  especially  numerous  in  the  anterior  indentation  region. 
The  cells  are  embedded  within  the  supportive  cells  and  do  not  extend  to  the  me- 
soglea. The  nematocyst  is  large  and  occupies  the  upper  two-thirds  of  the  cell.  A 
modified  cilium  gives  rise  to  the  cnidocil  which  is  located  to  the  side  of  the  ne- 
matocyst. The  capsule  of  the  nematocyst  consists  of  an  outer  electron-dense  layer 


FIGURE  1.  Scanning  electron  micrograph  of  a  planula  of  Cassiopeia.  The  planula  has  a  distinct 
anterior  end  and  posterior  end  and  is  uniformly  ciliated.  Just  prior  to  attachment  and  metamorphosis, 
an  indentation  is  found  in  the  anterior  end  (arrow).  A  =  anterior;  P  =  posterior.  Bar  =  20  ^m. 

FIGURE  2.  Transverse  section  of  the  apical  regions  of  ectodermal  supportive  cells.  These  supportive 
cells  possess  a  single  cilium,  numerous  microvilli,  and  electron-dense  granules.  The  nucleus  of  the  cell 
is  centrally  located  and  contains  a  prominent  nucleolus.  PAS-positive  granules  and  glycogen  particles  are 
located  more  basally  in  these  cells.  C  =  cilium;  GL  =  glycogen  particles;  GR  =  granules;  MV  =  microvilli; 
N  =  nucleus  of  supportive  cell;  V  =  vacuoles;  Y  =  PAS-positive  granules.  Bar  =  2  ^m. 

FIGURE  3.  Cilium  of  ectodermal  supportive  cell.  Each  cilium  projects  directly  from  the  apical 
membrane  of  the  cell  without  a  concavity  (arrow).  The  ciliary  rootlet  extends  deep  into  the  cytoplasm 
of  the  cell.  CR  =  ciliary  rootlet;  D  =  desmosome.  Bar  =  1  ^m. 

FIGURE  4.  Basal  body  (BB),  accessory  basal  body  (ABB),  ciliary  rootlet  (CR),  and  plaque-like 
structure  (P)  of  a  supportive  cell.  Bar  =  1  ^m. 

FIGURE  5.  Terminal  web  beneath  the  apical  membrane  of  a  supportive  cell.  The  micronlaments 
of  the  terminal  web  insert  at  the  cell  junctions.  J  =  junction  between  cells;  TW  =  terminal  web. 
Bar  =  2  /urn. 


322 


CASSIOPEIA  FINE  STRUCTURE 


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V.  J.  MARTIN  AND  F.-S.  CHIA 


FIGURE  6.    Septate  desmosome  between  2  supportive  cells.  CR  =  ciliary  rootlet;  V  =  vacuole.  Bar 


=  0.5 

FIGURE  7.  Foot  processes  of  the  ectodermal  supportive  cells.  These  processes  insert  on  the  mesoglea 
and  contain  numerous  PAS-positive  granules  and  glycogen  particles.  EC  =  ectoderm;  EN  =  endoderm; 
FP  =  foot  process  of  supportive  cell;  GL  =  glycogen  particles;  MG  =  mesoglea;  Y  =  PAS-positive  granules. 
Bar  =  1 


CASSIOPEIA  FINE  STRUCTURE  325 

and  an  inner  electron-lucent  layer.  The  thread  of  the  nematocyst  bears  arms  and 
spines  and  is  coiled  around  a  heavily  barbed  shaft.  The  nucleus  of  the  cell  is  small 
and  basally  located.  A  well-developed  Golgi  apparatus  is  also  present  in  the  basal 
region  of  the  cell.  Endoplasmic  reticulum  is  scarce. 

Developing  nematoblasts  are  located  at  the  base  of  the  epidermis  among  the 
foot  processes  of  the  supportive  cells  (Fig.  1 1).  They  do  not  make  contact  with  the 
free  surface  of  the  ectoderm.  The  cytoplasm  of  these  developing  nematoblasts  con- 
tains endoplasmic  reticulum,  a  Golgi  complex,  and  a  developing  nematocyst. 

The  endoderm  is  composed  of  a  single  layer  of  columnar-shaped  cells  very 
similar  in  structure  to  the  supportive  cells  of  the  ectoderm  (Fig.  12).  These  cells 
insert  on  the  mesoglea  via  their  basal  ends.  PAS-positive  granules,  vacuoles,  glycogen 
particles,  and  electron-dense  granules  are  abundant  in  these  basal  regions.  Micro- 
filaments  are  not  detected  in  these  cells.  Apically  the  cells  bear  a  single  cilium 
surrounded  by  a  collar  of  microvilli  which  projects  into  a  forming  gastrovascular 
cavity  (Figs.  12  and  13).  The  nucleus  is  centrally  located  and  often  contains  a 
nucleolus.  Mitochondria,  endoplasmic  reticulum,  and  polysomes  are  found  through- 
out the  cytoplasm. 

Clusters  of  interstitial  cells  are  scattered  among  these  supportive  cells  of  the 
endoderm.  The  nucleus  of  each  interstitial  cell  is  round  and  contains  a  prominent 
nucleolus.  Numerous  free  ribosomes  are  present  in  a  homogeneous  cytoplasm.  Other 
organelles  are  sparse  or  poorly  developed. 

A  thin  mesoglea  separates  the  ectoderm  from  the  endoderm.  The  mesoglea 
consists  of  a  meshwork  of  fibers  which  are  oriented  in  all  directions.  These  fibers 
are  embedded  within  a  PAS-positive,  amorphous  ground  substance. 

Examination  of  thick  plastic  serial  sections  and  comparable  thin  sections  re- 
peatedly demonstrate  the  absence  of  both  nerve  cells  and  glandular  cells  in  planulae 
of  Cassiopeia.  The  negative  PAS  staining  reaction  also  verifies  the  absence  of  glan- 
dular cells. 

DISCUSSION 

Results  from  this  study  and  that  of  Otto  (1978)  indicate  that  planulae  of  scy- 
phozoans  are  smaller  in  size  and  are  morphologically  simple  when  compared  to 
planulae  of  hydrozoans  and  anthozoans  (Table  I).  Planulae  of  Cassiopeia  and  Hal- 
iclystus  are  composed  of  only  4  cell  types,  whereas  the  hydrozoan  planulae  thus  far 
examined  contain  9  cell  types,  and  the  anthozoan  planulae  possess  anywhere  from 
9  to  1 5  cell  types.  In  Cassiopeia  the  planular  ectoderm  consists  of  1  type  of  supportive 
cell  and  1  type  of  nematocyte,  while  the  endoderm  contains  interstitial  cells  and  1 
kind  of  supportive  cell.  In  Haliclystus  3  types  of  cells  are  present  in  the  ectoderm 
( 1  form  of  supportive  cell,  1  form  of  nematocyte,  and  interstitial  cells),  and  only 
1  type  of  supportive  cell  comprises  the  endoderm.  Otto  (1978)  reported  microfila- 
ments  at  the  base  of  the  supportive  cells  in  both  ectoderm  and  endoderm  of  planulae 
of  Haliclystus.  In  planulae  of  Cassiopeia,  however,  microfilaments  were  found  only 

FIGURE  8.  Specialized  junctional  complexes  (arrows)  between  the  foot  processes  of  adjacent  sup- 
portive cells  and  between  the  foot  processes  and  the  mesoglea.  EC  =  ectoderm;  EN  =  endoderm; 
FP  =  foot  process  of  supportive  cell;  MG  =  mesoglea;  Y  =  PAS-positive  granules.  Bar  =  1  ^m. 

FIGURE  9.  Specialized  junctional  complex  between  2  foot  processes  of  the  supportive  cells.  These 
junctions  are  very  similar  to  desmosomes  in  that  the  unit  membranes  appear  thickened  due  to  the 
presence  of  a  dense  amorphous  layer  closely  applied  to  their  cytoplasmic  surfaces.  Microfilaments  (arrows) 
radiate  out  from  this  amorphous  substance.  A  slender  intermediate  dense  line  is  seen  in  the  middle  of 
the  intercellular  space  between  the  2  halves  of  the  junction.  Bar  =  0.5 


326 


V.  J.  MARTIN  AND  F.-S.  CHIA 


s  w/- 

:T\<    •  '  •'   ^WVrfV* 


FIGURE  10.  Nematocyte  at  the  ectodermal  surface  of  the  planula.  The  nematocyte  contains  a  large 
nematocyst  and  a  basally  located  nucleus.  A  well-developed  Golgi  body  is  usually  found  in  a  supranuclear 
position.  C  =  cilium;  G  =  Golgi  body;  N  =  nucleus.  Bar  =  2  ^m. 

FIGURE  1 1 .  Developing  nematoblast  located  at  the  base  of  the  ectoderm  among  the  foot  processes 
of  the  supportive  cells.  Specialized  junctions  between  the  foot  processes  of  the  supportive  cells  can  be 
seen  (arrows).  EC  =  ectoderm;  EN  =  endoderm;  MG  =  mesoglea;  NB  =  nematoblast.  Bar  =  1 


CASSIOPEIA  FINE  STRUCTURE  327 

in  the  apical  cytoplasm  and  the  foot  processes  of  the  ectodermal  supportive  cells. 
Both  the  planulae  of  Cassiopeia  and  Haliclystus  lack  glandular  cells  and  neural 
elements  which  generally  are  present  in  hydrozoan  and  anthozoan  planulae  (Lyons, 
1973a,  b;  Vandermeulen,  1974;  Chia  and  Crawford,  1977;  Martin  and  Thomas, 
1977,  1980;  Chia  and  Koss,  1979). 

Comparisons  of  the  ultrastructural  morphology  of  planulae  from  the  3  classes 
of  cnidarians  may  add  important  insights  into  the  phylogenetic  classification  of  the 
cnidarians.  Planulae  of  Pennaria  (Martin  and  Thomas,  1977,  1980)  and  Mitroco- 
mella  (Martin  et  al.,  unpublished  observations)  have  7  types  of  cells  in  the  ectoderm 
and  2  kinds  of  cells  in  the  endoderm.  These  hydrozoans  are  similar  to  the  scyphozoan 
planulae  in  that  in  both  classes  the  supportive  cells  of  the  ectoderm  and  the  endoderm 
are  arranged  in  a  simple  columnar  epithelium  with  basal  foot  processes  that  insert 
on  a  thin  mesoglea.  Also,  in  both  classes  the  planulae  contain  only  1  type  of  ne- 
matocyte.  The  2  classes  differ  in  that  the  hydrozoans  contain  neurosensory  cells, 
ganglionic  cells,  and  2  types  of  glandular  cells  in  the  ectoderm.  Anthozoan  planulae, 
when  compared  to  planulae  of  hydrozoans  and  scyphozoans,  tend  to  show  an  in- 
crease in  the  types  of  glandular  cells,  the  types  of  supportive  cells,  the  types  of 
nematocytes,  and  the  complexity  of  the  nervous  system.  The  ectoderm  of  anthozoan 
planulae  may  be  simple  columnar,  pseudostratified,  or  stratified  depending  upon 
the  species  examined  (Vandermeulen,  1974;  Chia  and  Crawford,  1977;  Lyons, 
1973a).  Planulae  of  Ptilosarcus  have  2  types  of  supportive  cells  and  3  types  of 
glandular  cells  in  the  ectoderm  (Chia  and  Crawford,  1977).  In  Pocillopora  and 
Balanophyllia  3  types  of  nematocytes  and  4  kinds  of  secretory  cells  are  found  in  the 
ectoderm  of  the  planulae  (Vandermeulen,  1974;  Lyons,  1973a,  b).  Planulae  of  An- 
thopleura  possess  3  types  of  glandular  cells  in  the  ectoderm,  and  they  exhibit  the 
most  complicated  nervous  system  described  to  date  for  a  planula  larva  (Chia  and 
Koss,  1979).  The  nervous  system  consists  of  an  apical  organ,  1  type  of  endodermal 
receptor  cell,  2  types  of  ectodermal  receptor  cells,  inter-neurons,  and  a  nerve  plexus. 

Werner  (1973),  in  his  analysis  of  the  evolution  of  the  cnidarian  classes,  proposed 
that  the  stem  form  of  the  recent  cnidarians  was  a  solitary,  sessile,  tetramerous  polyp. 
He  postulated  that  the  Anthozoa  were  an  early  offspring  from  this  common  ancestor, 
and  that  the  Scyphozoa,  Hydrozoa,  and  Cubozoa  arose  from  another  evolutionary 
line.  The  acceptance  of  Werner's  concept  would  result  in  the  classification  of  the 
phylum  Cnidaria  into  2  subphyla:  Anthozoa  and  Medusozoa.  In  the  Anthozoa  the 
polyp  is  the  sexual  adult  and  a  medusa  never  develops,  whereas,  in  the  Medusozoa 
a  medusa  is  the  normal  sexual  adult  and  the  polyp  is  regarded  as  a  larval  stage.  The 
Medusozoa  would  consist  of  the  extinct  class  Conulata  and  the  recent  classes  Scy- 
phozoa, Hydrozoa,  and  Cubozoa.  Based  on  the  comparative  fine-structural  mor- 
phology of  the  planulae  examined  to  date,  planulae  of  scyphozoans  and  hydrozoans 
appear  to  be  more  closely  related  to  each  other  than  are  planulae  of  the  scyphozoans 
and  anthozoans  or  planulae  of  the  hydrozoans  and  anthozoans  (Koss,  personal 
communication).  The  cells  which  comprise  hydrozoan  and  scyphozoan  planulae 

FIGURE  12.  Transverse  section  of  the  supportive  cells  of  the  endoderm.  Foot  processes  of  these 
cells  insert  on  the  mesoglea.  PAS-positive  granules,  vacuoles,  glycogen  particles,  and  electron-dense  gran- 
ules are  found  in  the  basal  regions  of  the  cells.  The  cells  possess  a  single  cilium  surrounded  by  microvilli 
which  projects  into  the  gastrovascular  cavity.  The  nucleus  of  the  cell  is  centrally  located.  C  =  cilium; 
EC  =  ectoderm;  GL  =  glycogen  particles;  MG  =  mesoglea;  N  =  nucleus  of  supportive  cell;  Y  =  PAS- 
positive  granules.  Bar  =  2  nm. 

FIGURE  13.  Transverse  section  of  the  gastrovascular  cavity  of  a  planula.  Numerous  cilia  and  mi- 
crovilli from  the  endodermal  supportive  cells  project  into  the  lumen  of  the  cavity.  GV  =  gastrovascular 
cavity;  MG  =  mesoglea.  Bar  =  5 


328  V.  J.  MARTIN  AND  F.-S.  CHIA 

are  morphologically  similar  and  are  not  as  complex  in  their  overall  structural  design 
as  are  the  cells  of  anthozoan  planulae.  Some  anthozoan  planulae  are  provided  with 
spirocysts  which  are  absent  in  planulae  of  hydrozoans  and  scyphozoans.  Further- 
more, many  anthozoan  planulae  possess  an  apical  organ.  Such  a  structure  has  not 
been  reported  in  a  hydrozoan  or  scyphozoan  planula. 

It  is  our  judgment  that  in  the  future  many  more  cnidarian  biologists  will  turn 
their  attention  to  the  comparative  cytology  of  planulae.  It  is  expected  that  results 
from  such  investigations  will  contribute  new  ideas  to  both  the  developmental  biology 
of  the  cnidarians  and  to  the  phylogenetic  classification  of  the  cnidarians. 

ACKNOWLEDGMENTS 

The  authors  wish  to  express  their  gratitude  to  Drs.  Sandra  Newell  and  Charles 
Cuttress  of  the  University  of  Puerto  Rico  for  providing  research  facilities.  We  thank 
Ron  Koss  and  Helen  Amerongen  for  critical  evaluation  of  the  manuscript.  This 
work  was  supported  by  a  grant  from  the  National  Research  Council  of  Canada. 

LITERATURE  CITED 

CHIA,  F.  S.,  AND  B.  CRAWFORD.  1977.  Comparative  fine-structural  studies  of  planulae  and  primary 

polyps  of  identical  age  of  the  sea  pen,  Ptilosarcus  gurneyi.  J.  Morphol.  151:  131-158. 
CHIA,  F.  S.,  AND  R.  Koss.  1979.  Fine-structural  studies  of  the  nervous  system  and  the  apical  organ  in 

the  planula  larva  of  the  sea  anemone  Anthopleura  elegant issima.  J.  Morphol.  160:  275-298. 
CLONEY,  R.  A.,  AND  E.  FLOREY.  1 968.  Ultrastructure  of  cephalopod  chromatophore  organs.  Z.  Zellforsch. 

89:  250-280. 
DUNLAP,  H.  L.  1966.  Oogenesis  in  Ctenophora.  Ph.D.  dissertation.  University  of  Washington,  Seattle, 

Washington. 
LANE,  B.  P.,  AND  D.  L.  EUROPA.  1965.  Differential  staining  of  ultrathin  sections  of  epon-embedded 

tissues  for  light  microscopy.  J.  Histochem.  Cytochem.  13:  579-582. 

LILLIE,  R.  D.  1954.  Histopathologic  technique  and  practical  histochemistry.  McGraw-Hill,  New  York. 
LUFT,  J.  H.  1961.  Improvements  in  epoxy  resin  embedding  methods.  J.  Biophvs.  Biochem.  Cytol.  9: 

409-414. 
LYONS,  K.  M.  1973a.  Collar  cells  in  planula  and  adult  tentacle  ectoderm  of  the  solitary  coral  Ba/anophyllia 

regia  (Anthozoa  Eupsammiidae).  Z.  Zellforsch.  145:  57-74. 
LYONS,  K.  M.  1973b.  Evolutionary  implications  of  collar  cell  ectoderm  in  a  coral  planula.  Nature  245: 

50-5 1 . 
MARTIN,  V.J.I  980.  The  role  of  the  interstitial  cell  during  embryonic  development  in  the  marine  hydrozoan 

Pennaria  tiarella.  Ph.D.  dissertation.  Wake  Forest  University,  Winston-Salem,  North  Carolina. 
MARTIN,  V.  J.,  AND  M.  B.  THOMAS.  1977.  A  fine-structural  study  of  embryonic  and  larval  development 

in  the  gymnoblastic  hydroid,  Pennaria  tiarella.  Biol.  Bull.  153:  198-218. 
MARTIN,  V.  J.,  AND  M.  B.  THOMAS.  1980.  Nerve  elements  in  the  planula  of  the  hydrozoan  Pennaria 

tiarella.  J.  Morphol.  166:  27-36. 
OTTO,  J.  J.  1978.  The  settlement  of  Haliclystus  planulae.  P.  13  in  Settlement  and  metamorphosis  of 

marine  invertebrate  larvae,  F.  S.  Chia  and  M.  E.  Rice,  Eds.  Elsevier,  New  York. 
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Reference:  Biol.  Bull.  163:  329-336.  (October.  1982) 


REGIONAL  DISTRIBUTION  OF  MUSCLE  FIBER  TYPES  IN  THE 
ASYMMETRIC  CLAWS  OF  CALIFORNIAN  SNAPPING  SHRIMP 

KATHLEEN  O'CONNOR,  PHILIP  J.  STEPHENS,  AND  JOHN  M.  LEFEROVICH 

Villanova  University,  Department  of  Biology,  Villanova,  PA  19085 

ABSTRACT 

The  properties  of  the  opener  and  closer  muscles  in  the  asymmetric  claws  of 
Alpheus  californiensis  have  been  investigated  using  sarcomere  length  measurements 
and  histochemical  techniques.  In  the  smaller  pincer  claw  two  types  of  muscle  fibers 
are  regionally  distributed  within  the  single  closer  muscle.  A  central  band  of  fibers 
have  short  (2.5  ^m)  sarcomeres  and  high  myofibrillar  ATPase  activity.  Intermediate- 
type  fibers  have  smaller  diameters,  sarcomeres  8.5  to  9  /urn  in  length  and  low  myo- 
fibrillar ATPase  activity.  The  snapper  closer  muscle,  by  contrast,  is  composed  of 
fibers  with  long  (11-14  /urn)  sarcomeres  and  low  myofibrillar  ATPase  activity. 
Opener  muscle  fibers  in  the  pincer  claw  have  shorter  sarcomere  lengths  than  their 
counterparts  in  the  snapper  claw. 

INTRODUCTION 

In  certain  crustaceans,  claw  dimorphism  is  accompanied  by  an  asymmetry  of 
claw  muscle  properties.  For  example,  in  lobsters  (Homarus  americanus)  the  rapidly 
closing  cutter  claw  has  a  large  proportion  of  fast  closer  muscle  fibers,  while  the 
slowly  closing  crusher  claw  is  composed  of  a  uniform  population  of  slow  muscle 
fibers  (Govind  and  Lang,  1974;  Lang  et  #/.,  1977).  In  addition  it  has  been  shown 
recently  that  a  similar  asymmetry  of  fiber  properties  is  present  between  the  claw 
opener  muscles  (Govind  et  al.,  1981). 

In  the  dimorphic  claws  of  snapping  shrimp  (Alpheus}  differences  exist  in  claw 
closer  muscle  properties  (Stephens  and  Mellon,  1979).  In  A.  heterochelis  and  A. 
armillatus  there  are  three  muscles  in  each  claw:  a  single  opener,  a  minor  closer,  and 
a  main  closer  muscle  (Ritzmann,  1974).  Analysis  of  sarcomere  lengths,  used  as  an 
indication  of  muscle  fiber  contraction  properties  (Atwood,  1973,  1976;  Josephson, 
1975),  reveals  that  differences  occur  only  in  the  main  closer  muscle.  In  the  larger 
snapper  claw  the  main  closer  muscle  is  composed  of  a  uniform  population  of  slow 
fibers  with  long  (10-15  ^m)  sarcomeres.  In  the  smaller  pincer  claw  the  main  closer 
muscle  has  two  populations  of  fiber  types.  Fast  fibers  in  the  central  portion  of  the 
main  closer  muscle  have  relatively  large  diameters  and  short  (2  and  3  ^m)  sarco- 
meres. Intermediate-type  muscle  fibers  have  sarcomeres  that  measure  between  6  and 
8  /urn  and  are  located  on  the  medial  and  lateral  margins  of  the  muscle. 

A  fascinating  feature  of  adult  snapping  shrimp  is  an  ability  to  reverse  claw 
configuration  (Wilson,  1903;  Przibram,  1931;  Mellon  and  Stephens,  1978).  Removal 
or  denervation  of  the  snapper  claw  causes  the  remaining  pincer  to  become  trans- 
formed into  a  new  snapper  claw,  while  a  pincer  regenerates  at  the  site  of  the  old 
snapper  claw.  Pincer-snapper  transformation  involves  a  change  in  the  properties 
of  the  main  closer  muscle  fibers  from  fast  and  intermediate  in  the  pincer  to  slow 
in  the  snapper  (Stephens  and  Mellon,  1979). 

Received  10  May  1982;  accepted  13  July  1982. 

329 


330  KATHLEEN  O'CONNOR  ET  AL. 

Measurement  of  sarcomere  length  is  one  method  used  to  examine  the  properties 
of  single  fibers  in  a  particular  muscle.  A  major  disadvantage  with  this  technique  is 
the  difficulty  in  constructing  a  complete  picture  of  the  properties  of  all  of  the  fibers 
in  a  given  muscle.  A  new  technique,  however,  allows  differentiation  of  fast  and  slow 
crustacean  muscle  fibers  based  on  histochemistry  (Ogonowski  and  Lang,  1979;  Sil- 
verman  and  Charlton,  1980).  In  a  previous  paper  we  showed  that  in  A.  californiensis 
the  sarcomere  lengths  of  the  single  claw  closer  muscle  are  similar  to  those  described 
for  the  main  closer  muscle  of  A.  heterochelis  (Stephens  et  #/.,  unpublished  obser- 
vations). In  the  present  study  we  have  used  histochemistry  and  sarcomere  length 
measurements  to  investigate  the  properties  of  the  closer  and  opener  muscles  in 
dimorphic  claws  of  A.  californiensis. 

MATERIALS  AND  METHODS 

Snapping  shrimp  (Alpheiis  californiensis}  were  obtained  commercially  from 
Venice,  California,  and  were  retained  individually  in  constantly  circulating,  artificial 
sea  water  at  14°C.  The  animals  were  fed  Tetramin  twice  weekly,  and  under  these 
conditions  lived  for  at  least  3  months  in  the  laboratory. 

Sarcomere  length  measurements 

Sarcomere  length  measurements  were  made  from  opener  or  closer  muscles  in 
pairs  of  claws  removed  from  adult  shrimp.  One  of  the  claw  muscles  was  carefully 
dissected  away  and  the  remaining  muscle  was  fixed  at  resting  length;  the  closer 
muscle  was  fixed  with  the  dactyl  in  the  open  position,  and  the  opener  muscle  with 
the  dactyl  in  the  closed  position  (Lang  et  #/.,  1977;  Stephens  and  Mellon,  1979). 
To  prevent  measurement  errors  due  to  muscle  contraction  (Meiss  and  Govind, 
1979),  the  dissected  claws  were  soaked  for  60  minutes  in  a  snapping  shrimp  saline 
in  which  calcium  ions  had  been  replaced  with  magnesium.  The  claws  were  then 
fixed  for  2  days  in  alcoholic  Bouin's  solution.  Individual  muscle  fibers  were  carefully 
teased  apart  in  80%  ethanol  on  a  microscope  slide,  and  examined  using  a  compound 
microscope  fitted  with  Normarski  optics.  The  length  of  five  successive  sarcomeres 
was  measured  using  a  calibrated  filar  ocular  micrometer.  At  least  three  measure- 
ments were  made  for  each  muscle  fiber  and  the  average  length  of  a  single  sarcomere 
was  calculated. 

Histochemistry 

Certain  histochemical  properties  of  the  muscle  fibers  in  the  dimorphic  claws 
were  examined.  Fully  developed  pincer  and  snapper  claws  were  removed  from  adult 
shrimp,  quickly  frozen  in  liquid  nitrogen,  and  sectioned  on  a  cryostat  microtome. 
Transverse  sections  ( 1 5  ^m  thick)  from  pairs  of  claws  were  mounted  on  glass  cover 
slips  and  the  myofibrillar  adenosinetriphosphatase  ( ATPase)  activity  was  determined 
using  a  technique  used  for  lobsters  (Ogonowski  and  Lang,  1979) — a  modification 
of  conventional  methods  for  vertebrate  tissue  (Padykula  and  Hermann,  1955).  A 
more  recently  published  technique  for  determining  myofibrillar  ATPase  activity 
(Silverman  and  Charlton,  1980)  was  employed  with  less  success. 

RESULTS 

Sarcomere  length  measurements 

Sarcomere  length  measurements  were  made  from  closer  and  opener  muscle 
fibers  in  pairs  of  fully  developed  claws  removed  from  the  same  animal.  The  closer 


ASYMMETRIC  CLAW  MUSCLE  FIBER  TYPES 


331 


B 


40 


(/> 

§      20 

is 


40 


20 


8.8.         13.1 


11.8 


8.5 


9 


13.1 


85       12.9 


25 


8 


8.9 1       13.1 


12.1 


8.7.         13.0 


12        16    0         4          8         12        16  0 

Sarcomere  length  (uM) 


12        16 


FIGURE  1 .  The  regional  distribution  of  sarcomere  lengths  in  the  claw  closer  muscle.  (A)  Diagram 
of  a  snapper  claw  with  the  propus  divided  into  9  regions.  (B)  Histograms  of  sarcomere  length  data  for 
closer  muscle  fibers  removed  from  each  region  (1-9)  in  a  pincer  (filled  columns)  and  snapper  (open 
columns)  claw  removed  from  the  same  animal.  Inset  numbers  represent  mean  sarcomere  length  values. 


muscle  was  divided  by  eye  into  9  regions  (Fig.  1A)  and  muscle  fibers  were  carefully 
removed  from  the  central  portion  of  each  region.  It  should  be  noted  that  observations 
were  not  made  from  fibers  in  region  3  since  only  opener  muscle  is  present  in  that 
region.  In  the  smaller  pincer  claw,  closer  muscle  fibers  located  in  the  dorsal  (1  and 
2)  and  ventral  (7  to  9)  regions  are  made  up  of  sarcomeres  with  mean  lengths  of  8.5 
to  9.0  nm  (Fig.  IB).  The  central  regions  (4  to  6),  by  contrast,  contain  muscle  fibers 
with  mostly  short  (2.5  nm)  sarcomeres.  A  similar  regional  distribution  of  different 
fiber  types  has  been  reported  for  the  pincer  main  closer  muscle  of  A.  arm  Hiatus 
(Stephens  and  Mellon,  1979). 

In  the  larger  snapper  claw,  the  closer  muscle  fibers  are  composed  of  long  sar- 
comeres (Fig.  IB).  Fibers  located  in  the  central  regions  of  the  muscle  have  sarcomere 
lengths  that  are  shorter  than  those  in  the  dorsal  and  ventral  regions,  however  these 
differences  are  not  statistically  different  (Student's  /-test). 


332 


KATHLEEN  O'CONNOR  ET  AL. 


30- 


J    20- 

15 

> 

a 

u> 
a 
O 

10- 


0-  - 


8 


10 


Sarcomere  length  (uM) 


FIGURE  2.  Histograms  to  show  the  sarcomere  lengths  of  opener  muscle  fibers  in  a  pair  of  pincer 
(filled  columns)  and  snapper  (open  columns)  claws  from  the  same  animal. 

Opener  muscle  fibers  from  pincer  and  snapper  claws  are  composed  of  sarcomeres 
of  different  lengths.  In  the  example  given  in  Figure  2,  opener  muscle  sarcomeres 
have  mean  lengths  of  7.5  /um  and  5.0  ^m,  respectively,  for  the  snapper  and  pincer. 
No  regional  differences  in  sarcomere  length  were  observed  in  the  opener  muscle  of 
either  claw. 

Histochemistry 

Figure  3  shows  photomicrographs  of  frozen  transverse  sections  taken  from  a 
pair  of  claws  and  stained  for  myofibrillar  ATPase  activity.  Sections  of  snapper  claws 
showed  uniform  light  staining  profiles  for  opener  and  closer  muscle  fibers  (Fig.  3C). 
In  sections  of  the  pincer  claw,  however,  a  central  band  of  closer  muscle  fibers  was 
always  darkly  stained  (Figs.  3D-F),  indicating  a  higher  ATPase  activity  in  these 
fibers  than  in  those  located  in  the  dorsal  and  ventral  regions.  Using  this  same  tech- 
nique on  lobsters,  Ogonowski  and  Lang  (1979)  showed  that  muscle  fibers  with  high 
myofibrillar  ATPase  activity  are  rapidly  contracting,  fast  muscle  fibers.  The  location 
of  the  dark-staining  closer  muscle  fibers  in  the  pincer  claw  (Fig.  3)  correlates  well 
with  the  location  of  short  sarcomere  fibers  (Fig.  1),  indicating  that  there  is  a  central 
band  of  fast  fibers.  In  the  pincer  claw  of  A.  armillatus  the  fast  main  closer  muscle 
fibers  have  a  larger  diameter  than  the  intermediate  muscle  fibers  (Stephens  and 
Mellon,  1979).  In  the  present  study  myofibrillar  ATPase  activity  was  used  to  dif- 
ferentiate between  fast  and  intermediate  fibers  in  the  pincer  closer  muscle.  Figure 
4  shows  closer  muscle  fiber  diameter  data  for  the  light-  and  dark-staining  fibers  in 
transverse  sections  of  a  pincer  claw,  and  for  closer  muscle  fibers  in  the  contralateral 
snapper  claw.  Although  there  is  no  statistical  difference  between  the  diameters  of 
the  two  types  of  pincer  closer  muscle  fibers,  it  is  apparent  that  the  dark  staining 
fibers  in  the  central  region  of  the  claw  have  a  slightly  larger  diameter  than  the  light- 
staining  fibers  in  the  ventral  and  dorsal  regions.  Furthermore,  the  closer  muscle 
fibers  in  the  snapper  are  about  twice  the  diameter  of  their  counterparts  in  the 
pincer  claw. 


DISCUSSION 

In  many  crustacean  neuromuscular  preparations  there  is  a  correlation  between 
the  speed  of  muscle  contraction,  sarcomere  length  (Atwood,  1973,  1976;  Govind 


ASYMMETRIC  CLAW  MUSCLE  FIBER  TYPES 


333 


FIGURE  3.    Myofibrillar  ATPase  activity  of  the  claw  muscles. 

(A,B):  Diagrams  of  a  snapper  (A)  and  pincer  (B)  claw  showing  the  locations  of  the  opener  (O)  and 
closer  (Cl)  muscles.  The  dark  band  in  the  pincer  closer  muscle  represents  the  location  of  the  fibers  with 
high  myofibrillar  ATPase  activity. 

(C-F):  Myofibrillar  ATPase  activity  of  the  claw  muscles  in  frozen  transverse  sections  (15  ^m  thick) 
of  a  snapper  (C)  and  a  pincer  (D-F)  claw.  A  band  of  fibers  with  high  myofibrillar  ATPase  activity  is 
present  in  the  pincer  closer  muscle.  Sections  D,  E,  F  were  taken  distally,  centrally,  and  proximally, 
respectively,  through  the  propus  of  the  pincer. 

Calibration:  1mm  (A,B)  and  500  ^m  (C-F). 


334 


KATHLEEN  O'CONNOR  ET  AL. 


B 


160          200          240          280          320 
Diameter    uM 

FIGURE  4.  The  diameter  of  closer  muscle  fibers  in  a  pincer  (A,  B)  and  snapper  (C)  claw.  Mea- 
surements were  made  from  frozen  transverse  sections  of  a  pair  of  claws.  Data  is  given  for  pincer  fibers 
with  low  ATPase  activity  (A),  high  ATPase  activity  (B),  and  for  snapper  closer  muscle  fibers. 


and  Lang,  1 974;  Josephson,  1 975)  and  myofibrillar  ATPase  activity  (Ogonowski 
and  Lang,  1979;  Ogonowski  el  a/.,  1980;  Silverman  and  Charlton,  1980).  Rapidly 
contracting  muscle  fibers  have  short  sarcomeres  and  high  myofibrillar  ATPase  ac- 
tivity, while  slow  muscle  fibers  have  long  sarcomeres  and  low  myofibrillar  ATPase 
activity.  Thus  in  the  pincer  claw  of  A.  californiensis,  the  fibers  in  the  central  region 
of  the  closer  muscle  are  presumably  fast,  while  those  located  on  the  dorsal  and 
ventral  surfaces  of  the  closer  muscle  are  presumably  intermediate  speed  fibers  (Figs. 
1  and  3).  A  histological  examination  of  fixed  claws  from  A.  armillatus  revealed 
similar  results  for  the  pincer  main  closer  muscle  (Stephens  and  Mellon,  1979).  In 
addition,  it  was  shown  that  the  centrally  located  fast  muscle  fibers  have  a  larger 
diameter  than  the  intermediate  fibers.  The  possibility  that  these  centrally  located 
muscle  fibers  contracted  immediately  prior  to  fixation,  producing  a  decreased  sar- 
comere  length  and  an  increased  fiber  diameter,  could  have  produced  erroneous 
results  (C.  Phillips,  personal  communication).  However  the  present  investigation, 
using  frozen  sections  and  also  prolonged  soaking  in  calcium-free  saline  prior  to 
fixation  to  prevent  muscle  contraction,  produced  a  similar  regional  distribution  of 
closer  muscle  fiber  types,  without  major  differences  in  muscle  fiber  diameter.  Fur- 
thermore, we  have  taken  transverse  sections  of  claws  of  A.  californiensis  following 
the  procedure  of  Stephens  and  Mellon  (1979)  and  have  observed  no  clear  regional 
differences  in  the  diameter  of  pincer  closer  muscle  fibers  (unpublished  observations). 
Moreover  it  is  interesting  that  a  distinct  band  of  fast  muscle  fibers  has  been  found 
in  the  central  region  of  the  closer  muscles  of  both  claws  of  larval  homarid  lobsters 
(Ogonowski  et  al.,  1980).  During  normal  development  the  closer  muscle  of  the  larger 
crusher  claw  becomes  uniformly  slow,  while  the  cutter  claw  closer  muscle  retains 
the  dimorphism  of  fiber  types  in  the  adult  (Lang  et  al.,  1977). 


ASYMMETRIC  CLAW  MUSCLE  FIBER  TYPES  335 

In  the  absence  of  direct  measurements,  histochemical  and  histological  properties 
of  muscle  fibers  can  provide  an  indication  of  contraction  speed.  In  many  crustacean 
muscles,  short  sarcomere  fibers  with  high  myofibrillar  ATPase  activity  are  fast,  while 
long  sarcomere  fibers  with  low  ATPase  activity  are  slow  (Atwood,  1973,  1976; 
Josephson,  1975;  Ogonowski  and  Lang,  1979;  Silvermann  and  Charlton,  1980). 
From  this  evidence  it  appears  that  the  pincer  closer  muscle  is  composed  of  fast  and 
intermediate  fibers,  while  the  snapper  closer  muscle  consists  of  fibers  that  contract 
slowly  but  produce  large  amounts  of  tension.  This  is  consistent  with  behavioral 
observations  made  on  snapping  shrimp  (Ritzmann,  1974;  Schein,  1975).  The  pincer 
claw  is  used  for  manipulation  of  small  objects  while  the  snapper  claw  is  used  only 
during  territorial  encounters  with  conspecific  shrimp.  The  dactyl  initially  moves  to 
open  the  snapper  claw  and,  in  Californian  snapping  shrimp,  a  pair  of  discs  on  the 
propus  and  the  dactyl  become  opposed  (Ritzmann,  1973).  The  closer  muscle  then 
develops  tension  to  overcome  the  adhesive  force  between  the  discs.  The  dactyl 
rapidly  closes  and  causes  a  jet  of  water  to  be  projected  towards  the  intruder  and 
also  produces  the  characteristic  snapping  sound. 

The  sarcomere  length  values  for  the  single  snapper  closer  muscle  of  A.  califor- 
niensis  (Fig.  IB)  are  similar  to  those  reported  for  the  main  closer  muscle  of  A. 
armillatus  (Stephens  and  Mellon,  1979).  Furthermore,  examination  of  A.  armillatus 
with  claws  undergoing  pincer-snapper  transformation  revealed  that  the  fast  and 
intermediate  main  closer  muscle  fibers  in  the  pincer  change  to  slow  muscle  fibers 
during  this  normal  developmental  process.  If,  in  A.  californiensis,  the  differences  in 
the  properties  of  the  closer  muscle  fibers  in  pairs  of  claws  represent  the  changes  that 
take  place  as  a  pincer  transforms  into  a  new  snapper,  it  is  apparent  that  there  are 
similar  changes  in  the  closer  muscle  fiber  properties  in  the  two  species.  However, 
we  have  shown  recently  that  the  differences  in  motor  axon  synaptic  facilitation 
reported  for  A.  armillatus  (Stephens  and  Mellon,  1979)  are  not  present  in  A.  cali- 
forniensis  (Stephens  el  al.,  unpublished  observations).  Examination  of  facilitation, 
using  pairs  of  junctional  and  synaptic  potentials  evoked  by  stimulation  of  the  ex- 
citatory axon,  showed  no  facilitation  in  the  snapper  closer  muscle.  In  fact,  synaptic 
depression  was  recorded  at  short  intervals  (<100  ms).  These  data,  together  with  the 
observation  that  the  closer  muscle  fibers  in  either  claw  appear  to  be  supplied  by  only 
one  excitor  axon,  has  raised  the  intriguing  possibility  that  claw  transformation  may 
involve  some  reorganization  of  peripheral  motor  axon  patterns,  as  seen  in  many 
vertebrate  preparations  (Rotshenker  and  McMahon,  1976;  Brown  and  Ironton, 
1977;  Hubel  el  #/.,  1977;  Jackson  and  Diamond,  1979;  Rotshenker  and 
Reichert,  1980). 

ACKNOWLEDGMENTS 

This  work  was  funded  by  grants  from  the  National  Science  Foundation  (BNS 
8113196)  and  the  Whitehall  Foundation.  The  authors  thank  Mr.  Ralph  Foy  and 
Mr.  Miles  Hermann  for  technical  assistance. 

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UREA  PARTHENOGENETICALLY  ACTIVATES  THE  CORTICAL 

REACTION  AND  ELONGATION  OF  MICROVILLI  IN  EGGS  OF  THE  SEA 

URCHIN,  STRONGYLOCENTROTUS  PURPURATUS 

HERBERT  SCHUEL,  PRAMILA  DANDEKAR1,  AND  REGINA  SCHUEL 

Department  of  Anatomical  Sciences,  SUNY  at  Buffalo,  Buffalo,  NY  14214,  and 
Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

ABSTRACT 

Isotonic  urea  is  believed  to  activate  sea  urchin  eggs  by  triggering  event(s)  that 
normally  follow  cortical  granule  secretion  at  fertilization,  particularly  surface  per- 
turbations that  result  in  elongation  of  microvilli  (Mazia  et  a!.,  1975).  However, 
Moser  (1940)  reported  that  urea  triggered  the  cortical  reaction.  Transmission  elec- 
tron microscopy  showed  that  unfertilized  Strongylocentrotus  purpuratus  eggs  dis- 
charge their  cortical  granules  in  isotonic  urea  (containing  1 .0  to  0. 1  mM  CaCl2  or 
25  mM  EGTA)  to  form  incipient  fertilization  envelopes  and  hyaline  layers.  These 
investments  quickly  disperse  in  urea.  Elongation  of  microvilli  follows  cortical  granule 
discharge.  Urea-activated  eggs  can  be  fertilized  after  return  to  sea  water  and  fail  to 
elevate  fertilization  envelopes  but  do  form  hyaline  layers.  Hyalin  must  be  secreted 
from  a  secondary  reservoir  in  these  eggs,  since  the  cortical  granule  store  is  discharged 
during  the  prior  urea  activation.  Cortical  granule  secretion  and  elongation  of  mi- 
crovilli do  not  occur  in  urea  plus  10  mMCaCU.  These  eggs  form  normal  fertilization 
envelopes  and  hyaline  layers  when  fertilized  after  return  to  sea  water.  Our  results 
show  that:  ( 1 )  urea  triggers  an  early  event  in  sea  urchin  egg  activation  that  stimulates 
cortical  granule  secretion;  (2)  cortical  granule  discharge  precedes  elongation  of  mi- 
crovilli in  urea-activated  eggs  as  it  does  during  normal  fertilization;  and  (3)  reduction 
or  removal  of  external  calcium  is  required  for  activation  by  urea. 

INTRODUCTION 

Parthenogenetic  agents,  including  isotonic  non-electrolytes  such  as  urea,  have 
been  used  to  study  the  sequence  of  events  and  causal  relationships  responsible  for 
the  activation  of  sea  urchin  eggs  during  fertilization  (reviewed  by:  Loeb,  1913;  Lillie, 
1919;  Allen,  1958;Epel,  1978;Schuel,  1978;Jaffe,  1980).  On  the  basis  of  a  scanning 
electron  microscopic  study  performed  on  Strongylocentrotus  purpuratus  and  Lyte- 
chinus  pictus,  it  was  suggested  that  urea  bypassed  the  cortical  reaction  and  activated 
the  sea  urchin  egg  by  releasing  a  represser  component  from  its  surface  (plasma 
membrane  and/or  vitelline  layer)  and  by  inducing  elongation  of  microvilli  (Mazia 
et  al.,  1975).  Ammonia  activation,  which  does  not  induce  the  cortical  reaction  (Loeb, 
1913;  Steinhardt  and  Mazia,  1973;  Epel  et  al.,  1974),  also  was  reported  to  promote 
elongation  of  microvilli  (Mazia  et  al.,  1975).  The  concept  advanced  by  Mazia's 
group  appeared  to  fit  with  observations  that:  ( 1 )  elongation  of  microvilli  normally 
occurs  subsequent  to  secretion  of  the  cortical  granules  during  fertilization  (Schroeder, 
1979);  (2)  detachment  of  the  vitelline  layer  from  the  egg's  plasma  membrane,  a  step 

Received  29  March  1982;  accepted  16  July  1982. 

1  Present  address:  Dept.  of  Obstetrics  and  Gynecology,  Univ.  of  Texas  Health  Sciences  Center, 
Houston,  TX  77030. 

337 


HERBERT  SCHUEL  ET  AL. 

in  the  assembly  of  the  fertilization  envelope  (Schuel,  1 978),  is  promoted  by  a  protease 
secreted  by  the  cortical  granules  (Longo  and  Schuel,  1973;  Schuel  et  al.,  1973; 
Vacquier  et  al.,  1973);  (3)  urea  removes  the  vitelline  layer  from  unfertilized  eggs 
(Moore,  1930);  and  (4)  ammonia  releases  a  surface  glycoprotein  that  results  in 
derepression  of  the  egg's  metabolism  as  normally  takes  place  during  fertilization 
(Johnson  and  Epel,  1975).  The  putative  role  of  the  release  of  represser  protein  from 
the  surface  of  ammonia-activated  egg  has  been  refuted  (Carroll  and  Epel,  1981). 
Moreover,  Mazia's  group  did  not  consider  the  possibility  that  urea  can  induce  the 
cortical  reaction,  and  that  elongation  of  micro villi  might  be  related  to  cortical  granule 
exocytosis  rather  than  to  surface  modifications.  Urea  had  previously  been  shown 
to  induce  secretion  of  the  cortical  granules  during  parthenogenetic  activation  of 
Arbacia  eggs  (Moser,  1940). 

The  present  study  was  undertaken  to  re-examine  the  effects  of  isotonic  urea  on 
the  surface  morphology  of  unfertilized  Strongylocentrotus  purpuratiis  eggs  by  means 
of  transmission  light  and  electron  microscopy.  A  preliminary  account  has  been 
presented  previously  (Schuel  and  Dandekar,  1981). 

MATERIALS  AND  METHODS 

Specimens  of  the  sea  urchin  Strongylocentrotus  purpuratiis  were  obtained  from 
Pacific  Bio-Marine  Laboratories  (Venice,  CA)  and  maintained  at  12-15°C  in  a 
marine  aquarium  (Aquarium  Systems,  Inc.,  Wickliffe,  OH).  Gametes  were  collected 
and  stored  as  described  previously  (Schuel  and  Schuel,  1981).  Only  batches  of  eggs 
that  yielded  95-100%  fertilization  in  test  insemination  (0.1  ml  eggs  per  5  ml  sea 
water  plus  0.1  ml  of  1%  sperm)  were  used  in  this  study.  Experimental  cultures  were 
incubated  at  15°C. 

Artificial  sea  water  was  prepared  from  Instant  Ocean  salt  mixture  (Aquarium 
Systems,  Inc.)  and  filtered  through  a  0.45/x  Millipore  filter.  Calcium-free  sea  water 
containing  25  mM  EGTA  (ethyleneglycol-bis(0-amino  ethyl  ether)N,N'-tetra  acetic 
acid)  was  prepared  according  to  Detering  et  al.  (1977).  Isotonic  urea  (1.0  M)  was 
prepared  in  deionized  water,  10  mM  CaCl2,  or  25  mM  EGTA  adjusted  to  pH  8.0 
with  NaOH.  EGTA  was  obtained  from  Sigma  Chemical  Co.,  St.  Louis,  MO. 

Unfertilized  eggs  were  activated  parthenogenetically  by  brief  exposure  to  isotonic 
urea  (Moser,  1940;  Mazia  et  al.,  1975).  Egg  suspensions  (1.0  ml)  were  added  to 
9.0  ml  of  urea  and  incubated  for  60  sec.  The  eggs  were  then  sedimented  by  gentle 
centrifugation  (IEC  Clinical  Centrifuge)  and  the  supernatant  discarded.  The  eggs 
were  resuspended  in  urea  (to  10  ml),  incubated  for  another  60  sec,  sedimented  again 
by  centrifugation,  and  finally  resuspended  in  sea  water.  This  treatment  took  about 
3  min.  After  exposure  to  the  urea  solutions,  the  eggs  were  inseminated  and  cultured 
in  sea  water.  In  some  experiments  eggs  were  observed  for  up  to  5  minutes  during 
a  single  continuous  treatment  with  9  parts  isotonic  urea  plus  1  part  sea  water. 

For  morphological  analysis,  eggs  were  fixed  with  3%  glutaraldehyde  in  sea  water. 
They  were  then  processed  for  examination  by  transmission  light  and  electron  mi- 
croscopy using  previously  described  procedures  (Longo  and  Anderson,  1972).  Thin 
sections  stained  with  uranyl  acetate  and  lead  citrate  were  examined  with  a  JEOL- 
100B  electron  microscope.  Thick  sections  stained  with  toluidine  blue  were  examined 
by  light  microscopy. 

RESULTS 

Live  Strongylocentrotus  eggs  were  observed  by  light  microscopy  during  and  fol- 
lowing treatment  with  isotonic  urea.  Thin  fertilization  envelopes  elevate  from  the 


UREA- ACTIVATED  EGGS  339 

surface  of  unfertilized  eggs  in  the  urea  solution.  Upon  continued  exposure  to  urea 
the  fertilization  envelopes  recede  toward  the  egg  surface  and  become  thinner,  until 
in  most  cases  no  vestige  of  the  fertilization  envelope  can  be  seen.  These  results  are 
consistent  with  previous  observations  that  urea  parthenogenetically  activates  the 
cortical  reaction  in  Arbacia  eggs  (Moser,  1940).  When  urea-activated  eggs  (two  60- 
sec  washes)  are  returned  to  sea  water  they  are  indistinguishable  from  control  eggs 
incubated  in  sea  water.  Urea-activated  eggs  can  be  fertilized.  However,  following 
insemination  none  of  these  eggs  lift  fertilization  envelopes,  but  most  form  hyaline 
layers.  Control  eggs  form  normal  fertilization  envelopes  and  hyaline  layers  upon 
fertilization.  Eggs  fertilized  following  urea  activation  divide  and  develop  at  the  same 
time  as  controls.  About  10%  of  the  urea-treated  eggs  fail  to  form  hyaline  layers  after 
subsequent  fertilization.  These  zygotes  divide  to  form  unorganized  grape-like  clusters 
of  blastomeres  during  cleavage.  These  findings  confirm  previous  observations  by 
Moore  (1930). 

The  effects  of  urea  treatment  on  the  surface  morphology  of  Strongylocentrotus 
eggs  was  determined  by  light  (data  not  shown)  and  electron  microscopic  (Figs.  1 
and  2)  analysis  of  fixed  and  sectioned  specimens.  Cortical  granules  are  located  sub- 
jacent to  the  plasma  membrane  in  unfertilized  (control)  eggs  (Fig.  1  A).  The  vitelline 
layer  is  attached  to  the  outer  surface  of  the  egg's  plasma  membrane,  and  short 
microvilli  are  present  at  the  egg  surface.  Upon  exposure  to  isotonic  urea  (9  parts 
plus  1  part  sea  water)  the  cortical  reaction  is  triggered  and  results  in  lifting  of  the 
fertilization  envelope  (Fig.  IB).  Patches  of  "hyalin-like"  material  are  seen  in  the 
perivitelline  space,  but  an  organized  hyaline  layer  does  not  form.  The  fertilization 
envelopes  begin  to  fragment  and  disperse  upon  continued  exposure  to  urea  (data 
not  shown).  Examination  of  eggs  returned  to  sea  water  after  two  60-sec  washes  in 
isotonic  urea  reveals  that  the  treatment  completely  removes  the  fertilization  enve- 
lopes (Fig.  2A).  Elongate  microvilli  are  prominent  features  at  the  surface  of  these 
eggs.  External  investments  (vitelline  layer/ fertilization  envelope  and  hyaline  layer) 
can  not  be  detected  outside  of  the  eggs.  After  these  eggs  are  fertilized,  they  form 
normal  hyaline  layers  but  do  not  form  fertilization  envelopes  (Fig.  2B). 

Several  other  aspects  of  the  responses  of  urea-activated  eggs  were  observed.  When 
eggs  are  suspended  in  urea  the  fertilization  envelope  appears  to  lift  simultaneously 
from  the  entire  circumference  of  the  activated  eggs.  Examination  of  fixed  and  sec- 
tioned specimens  indicates  that  in  each  individual  urea-activated  egg  the  cortical 
reaction  is  at  the  same  stage  around  the  entire  circumference  (data  not  shown).  By 
contrast,  during  normal  fertilization  the  cortical  reaction  and  the  elevation  of  the 
fertilization  envelope  start  at  the  site  of  attachment  of  the  fertilizing  sperm  and 
spread  around  the  surface  of  the  egg  (Moser,  1939a;  Anderson,  1968).  The  incidence 
of  eggs  in  the  population  that  show  a  cortical  reaction  increases  with  exposure  time 
to  urea  (Fig.  3).  The  data  are  presented  in  the  form  of  a  first  order  decay  plot  of 
unreacted  eggs  vs  exposure  time,  from  which  the  half  time  for  urea  activation  can 
be  estimated  to  be  about  90  sec. 

The  release  of  calcium  from  internal  stores  is  believed  to  play  a  critical  role  in 
the  initiation  of  cortical  granule  exocytosis  in  sea  urchin  eggs  during  fertilization 
and  upon  parthenogenetic  activation  (reviewed  by:  Epel,  1978;  Schuel,  1978;  Jaffe, 
1980).  Accordingly  we  studied  the  effects  of  calcium  on  the  parthenogenetic  induc- 
tion of  the  cortical  reaction  by  urea.  The  normal  calcium  concentration  of  sea  water 
is  10  mM  (Cavanaugh,  1964).  In  the  urea-activation  experiments  described  above 
(Fig.  2),  calcium  is  reduced  to  1.0  mM  in  the  first  wash  and  0.1  mM  in  the  second 
wash.  The  urea  solutions  used  by  Mazia's  group  (1975)  to  induce  elongation  of 
microvilli  contained  0. 1  mM  calcium.  We  found  that  when  25  mM  EGTA  is  added 


HERBERT  SCHUEL  ET  AL 


' 


FIGURE  1 .  Electron  micrographs  showing  parthenogenetic  induction  of  the  cortical  reaction  in 
unfertilized  Strongylocentrotus  eggs  by  isotonic  urea. 

A:  Control  egg  in  sea  water.  Cortical  granules  (CG)  located  just  below  the  egg's  plasma  membrane 
show  the  amorphous  (ac)  and  electron-dense  spiral  lamellae  components  characteristic  of  this  species. 


UREA-ACTIVATED  EGGS  341 

to  the  isotonic  urea,  cortical  granule  discharge  occurs  followed  by  elongation  of 
microvilli  as  described  above.  Control  eggs  incubated  under  similar  conditions  in 
calcium-free  sea  water  containing  25  mM  EGTA  do  not  show  a  cortical  reaction 
(data  not  shown).  Under  these  conditions  EGTA  reduces  the  free  calcium  in  the 
culture  solutions  to  below  10~7  M(Portzehl  et  a/.,  1964).  Conversely,  cortical  granule 
secretion  and  elongation  of  microvilli  do  not  take  place  when  unfertilized  eggs  are 
exposed  to  isotonic  urea  containing  10  mM  calcium  (Fig.  4A).  When  these  eggs  are 
fertilized  after  return  to  sea  water,  they  undergo  a  normal  cortical  reaction  to  produce 
fertilization  envelopes  and  hyaline  layers  (Fig.  4B).  The  thickened  tri-laminar  fer- 
tilization envelope  shows  sharp  "tent-like"  projections  indicative  of  structuralization 
by  secreted  cortical  granule  contents  in  Strongylocentrotus  ( Veron  et  #/.,  1 977;  Schuel 
et  al.,  1982).  These  observations  confirm  previous  findings  by  Moore  (1930)  that 
inclusion  of  calcium  in  the  urea  solutions  protects  the  egg's  capacity  to  form  a 
fertilization  envelope  upon  insemination  after  return  to  sea  water. 

DISCUSSION 

The  results  of  the  present  study  show  that,  contrary  to  previous  suggestions 
(Mazia  et  al.,  1975),  urea  does  not  mimic  the  presumed  effects  of  ammonia  in 
activating  sea  urchin  eggs  by  triggering  events  that  normally  occur  subsequent  to 
the  cortical  reaction.  Instead  urea  triggers  discharge  of  the  cortical  granules.  Fur- 
thermore, exocytosis  of  the  cortical  granules  precedes  elongation  of  microvilli  in 
urea-activated  eggs  just  as  it  does  during  normal  fertilization.  These  findings  confirm 
and  extend  earlier  observations  by  Moser  (1940).  Although  we  did  not  examine 
their  effects,  Moser  also  noted  that  other  non-electrolytes  (glycerol,  thiourea,  and 
sucrose)  elicited  the  same  kind  of  visible  cortical  response  as  urea. 

Elongation  of  microvilli  during  fertilization  or  upon  parthenogenetic  activation 
is  a  complex  process  that  depends  in  part  upon  the  insertion  of  the  limiting  mem- 
brane of  the  discharged  cortical  granules  into  the  egg's  original  plasma  membrane 
as  a  result  of  exocytosis  (Schroeder,  1979)  as  well  as  the  polymerization  of  actin  in 
the  cortex  to  form  bundles  of  microfilaments  (Burgess  and  Schroeder,  1977;  Carron 
and  Longo,  1982).  Urea  appears  to  mimic  other  parthenogenetic  treatments  such 
as  hypertonic  sea  water  (Sachs  and  Anderson,  1970)  and  calcium  ionophore  A23187 
(Chambers  and  Hinkley,  1979;  Carron  and  Longo,  1982)  which  induce  elongation 
of  microvilli  as  sequalae  to  cortical  granule  exocytosis.  Elongation  of  microvilli  in 
the  absence  of  cortical  granule  exocytosis  can  be  induced  by  application  of  hydro- 
static pressure  immediately  after  insemination  (Chase,  1967;  Hylander  and  Sum- 
mers, 1982)  and  by  treating  unfertilized  eggs  with  papain  (Spiegel  and  Spiegel,  1977). 
The  belief  that  ammonia  and  urea  induce  microvillar  elongation  in  the  absence  of 
prior  cortical  granule  exocytosis  (Mazia  et  al.,  1975)  appears  to  be  erroneous.  Other 
workers  who  examined  ammonia-activated  eggs  by  transmission  electron  micros- 
copy found  that  the  microvilli  do  not  elongate  and  the  cortical  granules  do  not 
secrete  (Nicotra  and  Arizzi,  1 979;  Hylander  and  Summers,  1981;  Carron  and  Longo, 
1982;  Schuel  and  Dandekar,  unpublished  data).  Cortical  granule  exocytosis  is  some- 
times seen  during  ammonia  activation  (Carroll  and  Epel,  1981).  However,  Mazia's 
group  (1975)  did  not  determine  whether  cortical  granule  exocytosis  had  occurred 
in  urea  or  ammonia  activated  eggs. 

The  vitelline  layer  (VL)  is  closely  applied  to  the  outer  surface  of  the  plasma  membrane.  Note  the  short 
microvilli  (MV).  Yolk  platelet  (Y).  50,OOOX. 

B:  Activated  egg  fixed  during  exposure  to  9  parts  isotonic  urea  and  1  part  sea  water.  The  cortical 
granules  have  discharged  and  a  thin  fertilization  envelope  (FE)  has  elevated  over  the  egg  surface.  A  patch 
of  "hyalin-like"  material  (H)  is  present  in  the  perivitelline  space.  Yolk  platelet  (Y).  33,300x. 


342 


HERBERT  SCHUEL  ET  AL. 


B 


FIGURE  2.  Electron  micrographs  showing  the  formation  of  the  hyaline  layer  in  urea-activated  eggs 
that  are  fertilized  after  return  to  sea  water.  33,300X. 

A:  Egg  washed  twice  with  isotonic  urea  and  fixed  immediately  after  return  to  sea  water.  Note  the 
numerous  elongate  microvilli  (MV)  and  the  absence  of  cortical  granules. 


UREA-ACTIVATED  EGGS 


343 


100 


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LU 

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20 


10 


100  200 

TIME  (sec) 


300 


FIGURE  3.  Effect  of  exposure  time  on  incidence  of  cortical  reaction  in  eggs  activated  by  urea.  The 
eggs  (1.0  ml)  were  exposed  to  9.0  ml  of  isotonic  urea  and  fixed  at  indicated  times.  The  incidence  of 
reacted  and  unreacted  eggs  was  scored  from  thick  sections  observed  by  light  microscopy. 

Morphological  (Endo,  1961;  Anderson,  1968)  and  biochemical  (Kane,  1970) 
observations  suggest  that  hyalin,  the  major  structural  protein  of  the  hyaline  layer 
(Stephens  and  Kane,  1970;  Citkowitz,  1971),  is  secreted  by  the  cortical  granules 
during  fertilization  (reviewed  by  Schuel,  1978).  In  addition  a  secondary  cytoplasmic 
reservoir  that  normally  is  slowly  released  during  embryogenesis  also  is  present  in 
unfertilized  eggs  (Kane,  1973).  These  concepts  have  become  controversial  because 
McBlaine  and  Carroll  (1980)  claimed  to  show  that  hyalin  is  a  cryptic  protein  on  the 
surface  of  unfertilized  eggs.  The  issue  has  been  resolved  by  recent  immunocyto- 
chemical  studies  using  monospecific  antibodies  against  pure  hyalin  (Hylander,  1981; 
Hylander  and  Summers,  1982;  McClay  and  Fink,  1982).  They  found  that  hyalin 
is  not  detectable  on  the  surface  of  eggs  prior  to  secretion  of  the  cortical  granules, 
and  is  sequestered  within  cortical  granules  of  unfertilized  eggs.  At  the  ultrastructural 
level  hyalin  is  localized  to  the  amorphous  component  of  Strongylocentrotus  cortical 
granules  (Hylander,  1981;  Hylander  and  Summers,  1982).  The  secondary  hyalin 
reservoir  is  stored  in  small  cytoplasmic  vesicles  (Hylander,  1981;  Hylander  and 
Summers,  1982).  In  the  present  study  the  hyaline  layer  formed  by  eggs  that  are 
fertilized  subsequent  to  urea  activation  must  have  been  secreted  by  the  secondary 
reservoir,  since  the  cortical  granule  store  was  discharged  and  dispersed  while  the 
eggs  were  being  pretreated  with  urea.  Hence  this  treatment  could  be  used  to  collect 
hyalin  from  its  two  cytoplasmic  reservoirs  for  further  study. 

Isotonic  urea  has  been  used  to  remove  the  vitelline  layer  from  unfertilized  sea 
urchin  eggs  (Moore,  1930)  and  the  soft  (non-cross-linked)  fertilization  envelope 
from  fertilized  eggs  prior  to  the  completion  of  hardening  (Schuel  et  al.,  1982).  When 
urea  is  applied  to  unfertilized  eggs,  it  induces  both  the  cortical  reaction  as  well  as 
the  dispersal  of  the  elevated  fertilization  envelope,  and  does  not  simply  remove  the 
vitelline  layer  as  previously  believed  (Mazia  et  al.,  1975).  Although  the  urea-activated 
egg  remains  receptive  to  sperm,  its  plasma  membrane  has  been  altered  by  cortical 


B:  Urea-activated  egg  that  was  fertilized  immediately  after  return  to  sea  water.  Fixed  10  min  after 
insemination.  Note  the  elongate  microvilli  (MV)  embedded  in  the  hyaline  layer  (HL)  that  invests  the  egg 
surface  and  the  absence  of  the  fertilization  envelope. 


344 


HERBERT  SCHUEL  ET  AL. 


MV 


FIGURE  4.    Absence  of  cortical  reaction  in  eggs  treated  with  isotonic  urea  containing  10  mM  CaCl2. 

A:  Egg  washed  twice  (60  sec  each)  with  isotonic  urea  containing  10  mA/CaCli,  and  fixed  immediately 

after  return  to  sea  water.  The  cortex  of  this  egg  is  identical  to  that  of  control  eggs  kept  in  sea  water 


UREA-ACTIVATED  EGGS  345 

granule  exocytosis  and  elongation  of  microvilli  to  resemble  that  of  a  naked  fertil- 
ized egg. 

The  effects  of  calcium  on  initiation  of  cortical  granule  secretion  by  urea  are 
paradoxical.  Certain  other  chemical  and  physical  treatments  that  parthenogeneti- 
cally  trigger  the  cortical  reaction  in  sea  urchins  require  external  calcium  (Moser, 
1939b).  Also,  the  release  of  calcium  from  an  internal  store  is  thought  to  be  part  of 
the  trigger  mechanism  for  cortical  granule  exocytosis  at  fertilization  or  partheno- 
genetic  activation  (Steinhardt  et  ai.,  1977;  Zucker  et  al.,  1978).  Calcium  is  stored 
at  several  sites  (vitelline  layer,  plasma  membrane,  limiting  membranes  of  cortical 
granules  and  other  cytoplasmic  organelles)  in  unfertilized  eggs  (Cardasis  et  al.,  1 978), 
although  the  identity  of  the  store  that  is  released  at  fertilization  is  unknown.  Urea 
appears  to  trigger  the  release  of  calcium  from  the  same  store  that  normally  is  released 
at  fertilization  (Zucker  et  «/.,  1978).  Yet  the  results  of  the  present  study  show  that 
urea  elicits  cortical  granule  secretion  only  when  the  external  calcium  is  reduced. 
Taken  together  these  findings  possibly  suggest  that  the  removal  of  calcium  from 
binding  sites  at  the  egg  surface,  perhaps  the  vitelline  layer  or  plasma  membrane, 
may  be  a  prerequisite  for  the  release  of  an  internal  store  to  trigger  exocytosis.  This 
feature  of  the  response  of  sea  urchin  eggs  to  urea  activation  may  provide  a  unique 
opportunity  to  study  the  initial  actions  of  calcium  in  stimulus-secretion  coupling 
and  activation  of  development.  Alternatively  it  is  possible  that  the  calcium  level  in 
normal  sea  water  renders  the  unfertilized  egg  impermeable  to  urea  and  thereby 
inhibits  parthenogenetic  activation  by  the  non-electrolyte.  Additional  work  is  re- 
quired to  answer  these  questions. 

ACKNOWLEDGMENTS 

Supported  by  grants  #PCM-77-14916A-02  and  PCM-82-01561  from  the 
National  Science  Foundation  to  H.S. 

We  wish  to  thank  Dr.  Don  P.  Wolf  and  Mr.  Jeffrey  Boldt  for  reading  this 
manuscript,  and  for  their  many  useful  suggestions. 

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Reference:  Biol.  Bull.  163:  348-354.  (October,  1982) 


EFFECT  OF  TEMPERATURE  AND  SALINITY  ON  LARVAL 

DEVELOPMENT  OF  SIBLING  SPECIES  OF  ECHINASTER 
(ECHINODERMATA:  ASTEROIDEA)  AND  THEIR  HYBRIDS 

STEPHEN  A.  WATTS,  R.  E.  SCHEIBLING,  ADAM  G.  MARSH,  AND  JAMES  B.  MCCLINTOCK 

Department  of  Biology,  University  of  South  Florida,  Tampa,  FL  33620 

ABSTRACT 

Adult  Echinaster  Type  1  and  Type  2  were  collected  along  the  west  coast  of 
Florida  (25 °C,  32%oS)  and  induced  to  spawn  in  the  laboratory.  Two-day  old  larvae 
of  Type  1,  Type  2,  and  their  hybrids  were  subjected  to  temperature  (T)  and  salinity 
(S)  combinations  (20,  25,  30°C;  25,  32,  39%oS).  Response  surface  isopleths  indicate 
that  Type  1  and  Type  2  larvae  exhibit  different  developmental  and  growth  rates  in 
response  to  T/S  combinations.  Salinity  was  a  dominant  factor  affecting  development 
and  growth.  High  and  low  salinities  inhibited  spine  development.  Developmental 
rates  were  directly  related  to  temperature.  Type  2  larvae  exhibited  a  greater  tolerance 
to  temperature  changes  than  did  Type  1 .  Hybrids  showed  intermediate  development 
and  growth  responses  at  the  apparent  optimal  conditions  and  exhibited  maternal 
characteristics. 

INTRODUCTION 

Sibling  species  (Campbell  and  Turner,  1979)  of  the  asteroid  Echinaster  occur 
in  the  vicinity  of  Tampa  Bay,  Florida.  One  is  blue-orange  in  color  and  the  other 
orange-brown.  Downey  (1973)  characterized  both  as  morphs  of  E.  modestus  based 
on  spination  in  dried  specimens.  However,  Campbell  and  Turner  (1979)  noted 
morphological  differences  between  the  two  and  concluded  that  they  were  sibling 
species.  Other  characteristics  of  these  species  are  reviewed  by  Scheibling  and  Law- 
rence (1982). 

The  species  have  been  classified  by  Atwood  (1973)  according  to  egg  type:  Type 
1  with  buoyant  eggs  and  larvae  which  are  planktonic  for  approximately  two  days, 
Type  2  with  demersal  eggs  and  completely  benthic  larvae.  Type  1  adults  generally 
spawn  in  the  spring  2-4  weeks  after  Type  2  (Scheibling  and  Lawrence,  1982).  Al- 
though these  Echinaster  species  are  found  in  different  habitats,  they  may  potentially 
interbreed. 

Phenotypic  variation  among  individuals  may  reflect  basic  genetic  differences  or 
environmentally  induced  modifications  and  phenotypic  plasticity  (Marcus,  1980). 
Genetic  differences  between  the  two  species  of  Echinaster  may  be  reflected  in  their 
physiology.  The  genetic  and  environmental  factors  that  control  the  growth  and 
development  of  the  species  may  be  distinguished  by  rearing  individuals  under  con- 
trolled labor  conditions. 

Hybridizatio  of  these  species  of  Echinaster  is  possible  by  cross-fertilization  in 
the  laboratory  (Scheibling,  1982),  although  there  is  no  evidence  that  hybridization 
of  the  two  morphs  occurs  in  nature.  Echinoderm  hybridization  between  both  genera 
and  species  has  been  documented  in  the  laboratory  (Tennent,  1910;  Harvey,  1956; 
Hagstrom  and  Lonning,  1961;  Hinegardner,  1967,  1975;  Horstadius,  1973;  Lucas 

Received  23  March  1982;  accepted  12  July  1982. 

348 


ECHINASTER  LARVAL  DEVELOPMENT  349 

and  Jones,  1976;  Strathmann,  1981)  and  in  the  field  (Verrill,  1909;  Hagstrom  and 
Lonning,  1961;  Strathmann,  1981).  Genetic  differences  may  be  reflected  by  differ- 
ential responses  of  the  species  and  their  hybrids  to  different  temperatures  and  sa- 
linities. The  purpose  of  this  study  was  to  examine  the  effects  of  temperature  and 
salinity  on  the  development  and  growth  of  sibling  species  of  Echinaster  and  their 
hybrids. 

MATERIALS  AND  METHODS 

Individuals  of  Echinaster  were  collected  in  May  and  June,  1981  from  the  coastal 
waters  of  the  eastern  Gulf  of  Mexico:  Type  1  from  the  Skyway  Bridge  at  the  mouth 
of  Tampa  Bay,  Type  2  from  the  intercoastal  waterway  at  Anna  Maria  Island.  In- 
dividuals were  maintained  in  the  laboratory  for  24  hours  in  aerated  sea  water  at 
field  temperature  and  salinity  of  25 °C  and  32%o  S.  Spawning  was  induced  with  0.001 
M  1-methyladenine  in  sea  water  either  in  vivo  via  intracoelomic  injection,  or  in  vitro 
using  excised  ovaries  and  testis.  Eggs  and  sperm  were  pooled  separately  from  3-6 
individuals.  Eggs  of  each  Type  were  both  fertilized  and  cross-fertilized  to  produce 
4  groups  of  zygotes:  Echinaster  Type  1  wild  (Type  19X3),  E.  Type  1  maternal 
hybrid  (Type  19  X  Type  23),  E.  Type  2  wild  (Type  29  X  3),  and  E.  Type  2  maternal 
hybrids  (Type  29  X  Type  13).  Eggs  of  each  morph  were  fertilized  less  than  two  weeks 
prior  to  spawning  in  nature.  The  lecithotrophic  embryos  were  maintained  at  field 
temperatures  and  salinities  during  early  cleavage  and  gastrulation.  After  two  days 
they  developed  into  motile,  modified  brachiolarian  larvae  and  were  placed  in  ex- 
perimental temperatures  and  salinities. 

Larvae  were  subjected  to  temperature  and  salinity  combinations  in  a  3  X  3  fac- 
torial design  (20,  25,  30°C;  25,  32,  39%o  S).  Ranges  of  temperatures  and  salinities 
experienced  by  Echinaster  in  the  field  are  10-33°C  and  25-35%o  S.  Preliminary 
experiments  indicated  that  larval  densities  of  5-25  individuals  per  bowl  had  no 
effect  on  development  and  growth.  Duplicate  sets  of  20  larvae  were  placed  in  6  cm 
diameter  glass  bowls  containing  approximately  25  ml  of  filtered  (0.45  /xm  Millipore 
filter)  sea  water  at  each  temperature  and  salinity  combination.  Only  one  set  of  Type 
2  hybrid  larvae  was  used.  Treatment  salinities  were  obtained  by  the  addition  of 
distilled  water  or  sea-salt  to  sea  water.  Temperature  and  salinity  were  controlled  in 
constant  environment  chambers  within  0.5°C  and  \%o  S  of  selected  values.  Larvae 
were  reared  in  the  dark  and  water  was  changed  daily.  Treatment  bowls  were  placed 
on  rotators  to  insure  adequate  mixing. 

The  developmental  stage  of  all  larvae  was  monitored  daily.  Data  represent  pooled 
observations.  The  following  stages  were  recorded:  brachiolarian  larvae,  first  ap- 
pearance of  the  first  and  second  pairs  of  tube  feet,  first  appearance  of  the  third  pair 
of  tube  feet,  first  appearance  of  the  fourth  pair  of  tube  feet,  and  the  appearance  of 
the  mouth.  All  stages  have  been  described  and  illustrated  (Kempf,  1966;  Atwood, 
1973).  In  Figures  1  and  2  data  represent  the  time  of  development  reached  by  75% 
of  all  individuals  in  a  T/S  combination.  Radii  of  larval  discs  were  measured  at 
several  stages.  Growth  was  found  to  be  linear  by  regression  analysis.  Average  growth 
was  measured  by  A  radius/A  time  (days)  X  100  (n  =  20). 

Response  surface  techniques  (see  Alderdice,  1972)  were  employed  for  statistical 
analysis  of  the  effects  of  temperature  and  salinity  combinations  on  larval  develop- 
ment and  growth.  Isopleths  were  determined  by  a  general  linear  model  contained 
in  the  Statistical  Analysis  System  (SAS).  Approximate  r2  values  between  0.81  and 
0.98  indicate  the  data  fit  this  model.  Isopleths  were  extrapolated  by  the  computer 
over  the  ranges  of  temperatures  and  salinities  tested. 


350 


STEPHEN  A.  WATTS  ET  AL. 


TYPE  1 


39 


TYPE  2 


25 


30   20 
TEMPERATURE  (°C) 


25 


30 


FIGURE  1.  Response  surface  estimates  of  time  (days)  until  the  appearance  of  the  first  and  second 
pairs  of  tube  feet.  Left  column  represents  EchinasterType  1  wild  (W)  and  hybrid  (H)  individuals.  Right 
column  represents  Type  2  wild  and  hybrid  individuals. 


RESULTS 

Response  surface  models  of  temperature  and  salinity  combinations  on  the  time 
(days)  until  the  appearance  of  the  first  and  second  pairs  of  larval  tube  feet  for 
EchinasterType  1,  Type  2,  and  their  hybrids  are  shown  in  Figure  1 .  The  appearance 
of  the  first  and  second  pairs  of  tube  feet  represent  an  early  stage  of  development  of 
the  larvae.  Type  2  wild  developed  faster  than  Type  1  wild.  The  apparent  optimal 
conditions,  i.e.,  those  resulting  in  the  fastest  rates  of  development,  are  not  different 
at  this  stage.  Small  changes  in  salinity  appear  to  have  a  pronounced  effect  on  de- 
velopment, indicated  by  narrow  isopleths.  Hybrids  show  intermediate  developmen- 
tal rates  at  the  apparent  optimal  conditions  with  the  maternal  influence  being  stron- 
gest. Hybrids  developed  more  slowly  than  the  maternal  parent  at  the  extreme  tem- 
perature and  salinity  combinations. 

Respon  j  surface  models  of  temperature  and  salinity  combinations  on  time 
(days)  until  ?  appearance  of  the  mouth  are  shown  in  Figure  2.  This  represents  a 
late  stage  in  tin  'evelopment  of  the  juvenile  just  prior  to  feeding.  The  mouth  of 
Echinaster  Type  1  wild  generally  appears  earlier  than  Type  2  wild.  Distinct  differ- 
ences were  seen  in  the  apparent  optimal  conditions.  Hybrids  generally  exhibit  a 
dominant  maternal  influence.  At  this  stage  Type  1  hybrids  exhibited  faster  rates  of 
development  than  the  maternal  parent,  and  Type  2  hybrid  rates  were  slower  than 
the  maternal  parent. 

Response  surface  models  of  temperature  and  salinity  combinations  on  growth 
rates  are  shown  in  Figure  3.  Growth  rates  were  faster  in  Type  2  individuals  and 


ECHINASTER  LARVAL  DEVELOPMENT 


351 


TYPE    1 


TYPE   2 


25 


25 


30      20 
TEMPERATURE   (°C) 


25 


30 


FIGURE  2.  Response  surface  estimates  of  time  (days)  until  the  appearance  of  the  mouth.  Left 
column  represents  EchinasterType  1  wild  (W)  and  hybrid  (H)  individuals.  Right  column  represents  Type 
2  wild  and  hybrid  individuals. 


correspond  with  faster  development  (Figures  1  and  2).  Apparent  optimal  conditions 
differed  between  Type  1  and  Type  2  wild.  Salinity  changes  had  a  pronounced  effect 
on  growth.  Isopleths  along  the  temperature  axis  indicated  the  eurythermality  of  both 
types,  with  Type  2  being  more  eurythermal  than  Type  1.  Maternal  hybrids  exhibited 
intermediate  growth  rates  at  the  optimal  temperature  and  salinity  combinations. 

Initial  exposure  of  the  larvae  to  T/S  combinations  induced  negligible  mortality 
in  all  combinations  (<  1%).  Greatest  mortality  (20%)  occurred  after  larvae  developed 
the  mouth,  and  was  presumed  to  result  from  lack  of  food.  There  was  no  higher 
mortality  among  hybrids. 

Abnormal  spine  development  was  observed  in  several  larvae  exposed  to  high 
and  low  salinities.  In  these  organisms  there  was  little  or  no  development  of  mouth- 
frame  armature  spines  or  of  the  terminal  spines  of  the  rays,  as  observed  by  light 
microscopy.  Several  larvae  exposed  to  high  salinity  exhibited  abnormal  ray  number 
development. 

DISCUSSION 

Temperature  and  salinity  are  of  primary  importance  in  determining  larval  de- 
velopment and  survival  in  marine  habitats.  The  importance  of  the  combined  effects 
of  temperature  and  salinity  has  been  emphasized  by  Kinne  (1970).  These  factors 
are  particularly  important  to  larvae  that  may  encounter  a  variable  environment,  as 
the  range  of  environmental  factors  tolerated  by  larvae  and  juveniles  is  usually  nar- 
rower than  that  tolerated  by  the  adult  (Vernberg  and  Vernberg,  1975). 


352 


STEPHEN  A.  WATTS  ET  AL. 


TYPE    1 


TYPE   2 


39 


30     20 
TEMPERATURE   (°C) 


25 


30 


FIGURE  3.  Response  surface  estimates  of  growth  rates.  Large  values  indicate  faster  rates  of  growth. 
Left  column  represents  Echinaster  Type  I  wild  (W)  and  hybrid  (H)  individuals.  Right  column  represents 
Type  2  wild  and  hybrid  individuals. 


Within  the  thermal  tolerance  limits  of  the  species,  warmer  temperatures  nor- 
mally accelerate  the  growth  processes.  Development  rates  of  larval  Echinaster  Types 
1  and  2  were  directly  related  to  temperature.  This  was  expected,  as  metabolic  rate 
increases  with  temperature  in  larvae  of  marine  invertebrates  in  general 
(Kinne,  1970). 

Salinity  greatly  influenced  development  in  Echinaster  Types  1  and  2.  Optimal 
development  and  growth  of  both  Types  1  and  2  occurred  at  28-32%o  S.  In  both 
types  poor  spine  development  occurred  in  many  individuals  exposed  to  low  and 
high  salinities.  In  many  individuals  there  was  little  or  no  development  of  the  mouth- 
frame  armature  spines  or  of  the  terminal  spines  of  the  rays.  This  indicates  that 
salinity  may  affect  the  deposition  of  carbonate  material  during  spine  formation. 
Metabolism  associated  with  carbonate  deposition  may  be  sensitive  to  changing  sa- 
linities and  not  due  to  the  concentrations  of  Ca++  and  Mg++  in  the  water,  as  poor 
spine  formation  occurred  at  both  low  and  high  salinities.  In  addition,  several  larvae 
exposed  to  high  salinities  exhibited  abnormal  ray  number  development,  i.e.,  3-  or 
4-rayed  instead  of  the  usual  5  (Watts  et  «/.,  1983). 

The  first  and  second  tube  feet  pairs  developed  earlier  in  Type  2  wild  individuals 
than  Type  1  individuals.  This  may  be  adaptive  to  Type  2  larvae  which  generally 
inhabit  shallow-water  seagrass  beds  subjected  to  wave  action.  Apparent  optimal 
temperatures  and  salinities  between  Type  1  and  2  do  not  differ  at  this  early  stage 
of  development.  However,  low  temperature  has  a  more  pronounced  effect  on  Type 
1  than  Type  2  as  indicated  by  slower  developmental  rates  in  Type  1  larvae. 


ECHIN ASTER  LARVAL  DEVELOPMENT  353 

The  mouth  generally  developed  earlier  in  Type  1  wild  individuals  than  in  Type 
2.  The  mouth  in  Type  1  appears  after  the  third  pair  of  tube  feet  while  the  mouth 
in  Type  2  does  not  appear  until  after  the  fourth  pair  of  tube  feet.  The  differences 
in  developmental  rates  are  similar  to  those  found  by  Atwood  (1973)  and  Kempf 
(1966).  Distinct  differences  were  seen  in  the  apparent  optimal  temperatures  and 
salinities  at  this  stage.  At  high  temperatures,  Type  2  individuals  developed  faster 
than  did  Type  1,  and  these  differences  are  also  shown  in  faster  growth  rates  of  Type 
2  larvae.  However,  once  the  mouth  is  present  and  the  juveniles  begin  to  feed,  Type 

1  grows  more  rapidly  than  Type  2  (Scheibling,  1982). 

Type  2  appears  to  be  more  eurythermal  than  Type  1  and  this  may  be  reflective 
of  its  shallow  water  existence.  Type  1  generally  inhabits  deeper  water  than  Type  2 
(Atwood,  1973).  The  greater  tolerance  of  Type  2  for  a  wider  range  of  temperatures 
in  shallow  water  may  be  related  to  past  selective  influences  by  the  thermal  envi- 
ronment on  previous  generations.  Larval  development  and  growth  rates  may  also 
be  influenced  by  environmental  conditions  experienced  by  the  adults,  particularly 
during  gonadal  development  (Davies,  1958).  Thermal  tolerance  of  aquatic  poikil- 
otherms  is  in  part  dependent  on  their  thermal  history  (Kinne,  1970). 

Hybrids  of  Echinaster  Type  1  and  Type  2  generally  exhibited  intermediate  de- 
velopmental and  growth  rates  at  the  apparent  optimal  temperatures  and  salinities. 
Type  1  hybrids  at  time  of  the  appearance  of  the  mouth  exhibited  faster  develop- 
mental and  growth  rates  than  the  maternal  parent,  while  Type  2  hybrids  exhibited 
slower  developmental  and  growth  rates  than  their  maternal  parent.  This  indicates 
that  developmental  and  growth  rates  are  directly  related,  but  separate  phenomena. 

These  factors  suggest  that  the  differences  in  temperature  and  salinity  responses 
between  the  two  morphs  are  genetically  controlled,  and  are  not  environmentally 
influenced.  The  maternal  influence  is  apparently  stronger  in  the  hybrids  than  the 
paternal  influence.  Andronikov  (1967)  found  that  the  zygote  obtained  by  fertilization 
of  Strongylocenlrotus  nudus  ova  via  S.  intermedius  spermatozoa  showed  the  same 
level  of  heat  tolerance  as  a  normal  S.  nudus  zygote,  indicating  the  dominant  influence 
of  the  eggs'  thermal  characteristics.  Hinegardner  (1975)  crossed  the  sand  dollars 
Encope  californicus  and  Dendraster  excentricus  and  reported  that  during  accidental 
exposure  of  larvae  to  high  temperatures,  physiological  responses  to  stress  were  in- 
herent in  the  hybrids.  Furthermore,  Hinegardner  found  that  the  hybrids  exhibited 
paternal  characteristics,  which  is  considered  to  be  a  general  tendency  of  echinoderm 
hybrids.  Lucas  and  Jones  (1976)  found  that  hybrids  of  the  asteroids  Acanthaster 
planci  and  A.  brevispinus  were  morphologically  intermediate.  Marcus  (1980)  also 
found  intermediate  differences  indicating  joint  influences  of  maternal  and  paternal 
genomes  in  the  echinoid  Arbacia  punctulata  from  two  geographic  localities.  Schei- 
bling (pers.  obs.)  found  that  Type  2  hybrids  reared  in  the  laboratory  morphologically 
resemble  the  maternal  parent.  Cytoplasmic  constituents  in  the  eggs  may  influence 
the  developing  larvae  in  Echinaster.  The  genetic  influence  of  the  maternal  and 
paternal  genomes  is  apparently  variable  in  echinoderms. 

The  differences  in  the  development  and  growth  of  the  sibling  species  of  Echi- 
naster appear  to  be  genetically  controlled,  and  not  the  result  of  environmentally 
induced  modifications  of  their  physiology.  Although  most  echinoderm  hybrids  do 
not  live  beyond  the  larval  stage  (Hinegardner,  1975),  Scheibling  (1982)  reared  Type 

2  hybrids  for  one  year  in  the  laboratory  and  found  their  growth  rates  to  either  equal 
or  exceed  that  of  Type  2  wild  individuals.  The  high  degree  of  genetic  compatability 
between  the  two  species,  as  demonstrated  by  the  FI  hybrids,  indicate  that  the  two 
species  are  closely  related  and  suggests  recent  speciation. 


354  STEPHEN  A.  WATTS  ET  AL. 

ACKNOWLEDGMENTS 

We  would  like  to  thank  Dr.  John  M.  Lawrence  for  discussions  on  the  manuscript. 
We  also  thank  Drs.  S.  Bell,  D.  Mermer,  J.  Simon,  and  C.  Dawes  for  assistance. 

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Wilt  and  N.  K.  Wessels,  Eds.  T.  Y.  Cromwell  Co.,  New  York. 
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ABSTRACTS  OF  PAPERS  PRESENTED  AT  THE  GENERAL  SCIENTIFIC 
MEETINGS  OF  THE  MARINE  BIOLOGICAL  LABORATORY 

AUGUST  17-20,  1982 

Abstracts  are  arranged  alphabetically  by  first  author  within  the  following  categories: 
actin,  microtubules,  membrane  transport,  and  microanatomy;  ecology;  fertilization 
and  development;  neurobiology;  parasitology  and  pathology;  photoreceptors;  and 
physiology  and  biophysics.  Author  and  subject  references  will  be  found  in  the  regular 
volume  index  in  the  December  issue. 

ACTIN,  MICROTUBULES,  MEMBRANE  TRANSPORT, 
AND  MICROANATOMY 

Glutamate  dehydrogenase  activity  in  wood-  and  mud-burrowing  bivalve  molluscs. 
CRAIG  J.  ANMUTH  (Oberlin  College),  S.  M.  GALLAGER,  R.  MANN,  AND  R.  S. 
ALBERTE. 

Glutamate  dehydrogenase  (GDH)  catalyzes  the  interconversion  of  «-ketoglutarate  and  L-glutamate: 
a-ketoglutarate  +  NH/  +  NADH  or  NADPH  ^  L-glutamate  +  NAD+  or  NADP+  +  H2O.  In  animal 
tissue  this  enzyme  is  localized  in  mitochondria  and  uses  either  NADH  or  NADPH.  The  bacterial  enzyme, 
however,  is  NADPH  specific.  We  examined  the  energy  substrate  specificity  and  activity  of  GDH  and 
ammonia  uptake  or  excretion  in  the  wood-boring  bivalves  (shipworms)  Lyrodus  pedicellatus,  Bankia 
gouldi,  and  Teredo  navalis,  and  the  mud-burrowing  clam  Solemya  velum.  All  these  animals  possess 
symbiotic  bacteria  (unpublished  data). 

Animals  were  maintained  in  ambient  sea  water  (ammonia  concentration  2  ^mole).  The  shipworms 
were  dissected  into  gill  tissue,  tissue  encompassed  by  the  valves,  and  remaining  tissues,  while  S.  velum 
was  dissected  into  gill  tissue  and  remaining  tissue.  The  tissues  were  homogenized,  subcellular  components 
collected  by  differential  centrifugation,  and  the  pellets  lysed  to  release  GDH.  The  reaction  was  assayed 
in  the  forward  direction  by  the  oxidation  of  NADH  or  NADPH.  Whole  animal  NADH-dependent  GDH 
activities  were  156.86  ±  13.0  (s.e.;  N  =  3),  123.81  ±  32.1  (N  =  3),  49.43  (N  =  1)  72.53  ±  13.5  (N  =  3) 
mA/  NADH/g  wet  weight/h  for  L.  pedicellatus,  B.  gouldi,  T.  navalis,  and  S.  velum,  respectively;  while 
the  NADPH-dependent  activities  were  generally  higher:  195.11  ±52.1,  157. 84  ±69.9,  41.23,  122.46 
±  46.9  mA/  NADPH/g  wet  weight/h,  respectively.  Activity  was  greatest  in  the  gill  tissues.  Prior  long  term 
incubation  of  animals  at  50  ^mole  ammonia  concentrations  reduced  GDH  activity. 

A  significant  portion  of  NADPH-dependent  GDH  activity  could  be  bacterial  in  origin.  High 
NADPH-specific  GDH  activity  was  found  in  a  monoxenic  culture  of  bacteria  isolated  from  the  gill  tissue 
of  L.  pedicellatus.  All  shipworm  species  exhibited  net  ammonia  rates  of  1-2  jtmole  NH4+/g  wet  weight/ 
h  for  intact  animals  at  ambient  ammonia  concentrations.  In  contrast  S.  velum  exhibited  net  ammonia 
excretion.  It  is  possible  that  the  symbiotic  bacteria  present  can  account  for  a  significant  portion  of  the 
ammonia  uptake  by  L.  pedicellatus  and  contribute  to  its  nitrogen  metabolism. 

We  acknowledge  the  support  of  ONR,  Contract  No.  N00014-79-C-0017NR083-004  and  the  M.B.L. 
Marine  Ecology  Course.  We  thank  J.  Waterbury,  C.B.  Calloway,  and  C.  Cavanaugh  for  use  of  un- 
published data. 

A  possible  role  of  protein  carboxymethylase  in  fertilization  and  sperm  motility. 
C.  ARANOW,  J.  COHN,  AND  W.  TROLL. 

The  evaluation  of  sperm  motility  is  an  important  factor  in  determining  the  cause  of  infertility.  Non- 
motile  or  poorly  motile  sperm  of  many  species  can  not  successfully  fertilize  eggs.  In  man,  low  sperm 
motility  has  been  shown  to  correlate  with  low  fertility  (Gagnon  el  al.  1982).  We  examined  protein 
carboxymethylase  (PCM),  an  enzyme  described  in  a  diversity  of  biologic  systems  such  as  hormone 
exocytosis,  bacterial  chemotaxis,  and  leukocyte  stimulation  and  migration.  Its  activity  has  been  observed 
in  the  membrane  fractions  of  motile  sperm.  PCM  catalyzes  the  methylation  of  free  carboxyl  groups  of 
protein  substrates  to  form  methyl  esters  using  S-adenosyl  methionine  (SAM)  as  a  methyl  donor.  The 
methyl  ester  bond  formed  in  this  reaction  is  hydrolyzed  either  spontaneously  at  neutral  or  alkaline  pH 

355 


356  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

or  enzymatically  by  a  protein  methylesterase  releasing  methanol.  We  assayed  for  PCM  by  incubating  sea 
water  with  sperm  and  tritium-labeled  methionine  and  determined  the  radioactive  methanol  extracted  by 
toluene.  The  sperm  incorporates  methionine  which  reacts  with  ATP  to  form  labeled  SAM.  Labeled 
methyl  groups  donated  in  the  PCM  reaction  hydrolyze  releasing  radioactive  methanol.  Shaking  the 
scintillation  vial  at  selected  times  extracts  the  methanol  generated  by  the  system  into  a  toluene  scintillent 
which  is  counted.  In  motile  sperm,  an  initial  rapid  accumulation  of  methanol  counts  released  by  the 
enzyme  system  plateaus  with  time.  We  examined  the  effect  of  H2O2  and  dithiothreotol  (DTT)  on  PCM 
since  H2O2  has  been  implicated  in  the  prevention  of  polyspermy  (the  penetration  of  the  egg  by  more 
than  one  sperm)  in  Arbacia  punctulata.  H2O2  decreases  both  the  fertility  and  motility  of  Arbacia  sperm 
while  DTT  reverses  these  effects.  We  now  report  that  H2O2  appears  to  inhibit  while  DTT  potentiates  the 
PCM  enzymatic  system.  Thus  the  mechanism  of  H2O2  inactivation  and  DTT  reactivation  of  Arbacia 
sperm  may  be  due  to  the  effect  of  these  compounds  on  PCM. 

Support  from  NIH  6060  to  W.  Troll  and  from  NYU  School  of  Medicine  Honors  Program  to  C. 
Aranow  and  J.  Cohen. 

Fine  structure  of  tissue  warming  the  brain  and  eye  in  tuna.  BARBARA  BLOCK  (Duke 
University),  EUGENE  COPELAND,  AND  FRANK  CAREY. 

The  gills  of  teleosts  have  a  total  surface  area  that  is  efficiently  arranged  to  obtain  gas  exchanges  by 
diffusion.  The  same  physical  structures  provide  very  efficient  heat  transfer  so  that  convection  cooling 
keeps  the  general  body  temperature  within  a  fraction  of  that  of  the  ambient  water.  Some  tissues,  usually 
those  associated  with  survival  capabilities,  have  developed  thermogenic  properties.  The  released  heat  is 
conserved  by  vascular  counter  current  heat  exchanges.  The  recent  discovery  of  thermogenic  tissue  as- 
sociated with  the  brain  of  swordfish  (Carey  1982,  Science  216:  1327-1329)  caused  us  to  examine  a 
similarly  located  tissue  in  bluefin  tuna  where  we  have  recorded  as  high  as  13.2°C  temperature  elevation 
in  the  brain.  A  hot  spot  was  located  in  pigmented  tissue  that  incorporates  a  counter  current  vascular 
supply  to  both  eye  and  brain.  Grossly,  the  tissue  was  opalescent  and  fatty  in  appearance  with  punctate 
localizations  of  melanin.  At  the  fine  structure  level  the  cells  (other  than  the  classical  melanocytes)  at  first 
glance  look  like  partially  compacted  leucocytes,  both  granular  and  agranular.  The  major  population  is 
granular  and  marked  by  having  droplets  in  various  stages  that  are  only  partially  osmiophilic  (i.e.  largely 
saturated  lipid?).  The  droplets  are  consistently  associated  with  varying  amounts  of  non-membrane  bound 
granular  material.  Occasionally,  a  very  large  osmiophilic  lipid  droplet  is  seen.  The  much  fewer  agranular 
cells  usually  have  a  dense  array  of  rough  endoplasmic  reticulum  filled  with  material.  None  of  the  cells 
showed  the  dense  concentration  of  mitochondria  seen  in  the  similarly  located  tissue  of  the  swordfish. 
Since  peroxisomes  are  known  to  catalyze  metabolic  reactions  in  such  a  way  that  heat  is  a  byproduct,  the 
tissues  were  checked  histochemically  with  a  diaminobenzidine  technique.  The  preliminary  results  were 
interesting  but  not  conclusive. 

Supported  by  an  award  from  Sigma  Xi  and  funds  from  the  Zoology  Department  of  Duke  University 
to  B.A.B. 

Temperature-induced  disassembly  of  isolated  marginal  bands  and  reassembly  of 
marginal  band  tubulin.  WILLIAM  D.  COHEN  (Hunter  College,  NY),  GEORGE  M. 
LANGFORD,  AND  ROGER  D.  SLOBODA. 

The  marginal  bands  (MBs)  of  microtubules  in  living  dogfish  erythrocytes  are  cold-labile,  and  reas- 
semble upon  rewarming.  However,  isolated  dogfish  erythrocyte  MBs  are  stable  at  0°C  in  a  medium 
consisting  of  1  mA/  MgCl2,  5  mA/  EGTA,  100  mA/  PIPES,  pH  6.8  (Cohen  el  al.  1982,  J.  Cell  Biol.  93: 
828-838).  Observations  by  Diana  Bartelt  (Ph.D.  Dissertation,  CUNY,  1982)  suggested  that  this  was  due 
to  insufficient  ionic  strength.  Using  darkfield  microscopy  in  the  present  work,  isolated  MBs  were  observed 
to  disassemble  at  0°C  in  30-60  minutes  if  1 50  mA/  KC1  was  included  in  the  medium.  To  test  for  tubulin 
reassembly,  GTP  was  added  to  1  mA/  and  samples  were  rewarmed  on  slides.  Rodlets  appeared  within 
a  few  minutes,  and  rubsequently  formed  masses  of  fibrillar  elements,  some  approaching  MBs  in  thickness. 
Reassembly  was  inhiS  jd  by  1  mA/  colchicine.  Formation  of  the  fibrillar  aggregates  appeared  to  involve 
rodlet  alignment  resin  •  from  flow,  and  the  aggregates  dispersed  into  individual  rodlets  after  vigorous 
tapping  on  the  coverslip.  Further  study  will  be  required  to  determine  whether  the  organization  of  the 
fibrillar  aggregates  resembles  that  of  MBs.  At  150  mA/  KC1,  reassembly  occurred  at  approximately  0.4 
mg  protein/ml,  but  not  at  0.2  mg/ml.  However,  reassembly  did  occur  at  0.2  mg/ml  in  15  mA/  KC1, 
showing  that  lower  salt  concentrations  favored  assembly.  Negative  staining  of  reassembled  material  re- 
vealed principally  normal-looking  microtubules,  some  of  which  adhered  to  others  along  their  length. 
After  centrifugation  of  0°C-disassembled  MB  preparations  at  100,000  X  g  for  30  min  (approx.  4°C), 
reassembly  occurred  as  before,  demonstrating  that  it  did  not  involve  the  presence  of  MB  microtubule 


ACTIN,  MICROTUBULES,  ETC.  357 

seeds.  Following  reassembly  spectrophotometrically  with  such  preparations,  absorbance  (350  or  400nm) 
rose  to  a  plateau  in  a  few  min  at  25°C.  SDS-PAGE  showed  that  the  reassembled  material  consisted  of 
tubulin  plus  smaller  amounts  of  two  low  molecular  weight  proteins  (LMWs).  Studies  are  in  progress  to 
characterize  further  MB  tubulin  and  to  determine  whether  the  LMWs  are  MB  microtubule-associated 
proteins  (MAPs). 

The  authors  thank  Dr.  Joel  L.  Rosenbaum  for  his  support  and  advice.  Supported  by  NSF  grant 
#PCM8 1-07 195  and  CUNY-PSC  grant  #14051  to  W.D.C. 

Lability  ofmitotic  spindle  microtubules  during  cell  lysis.  L.  CRESWELL,  T.  OTTER, 
D.  A.  LUTZ,  AND  S.  INOUE  (Marine  Biological  Laboratory). 

The  transition  ofmitotic  spindles  from  their  living  state  to  a  less  labile  in  vitro  or  "isolated"  condition 
was  studied  by  lysing  sand  dollar  (Echinarachnius  parma)  eggs  within  the  fertilization  envelope.  When 
eggs  are  lysed  in  PEM  buffer  (10  mA/  PIPES,  pH  6.8,  5  ra/V/  EGTA,  0.5  mA/  MgCl2),  the  birefringence 
(BR)  of  the  half-spindle  decays  to  5%  of  its  initial  value  within  10  minutes.  When  Triton  X-100  (0.1% 
or  1%)  is  included  in  the  PEM,  the  initial  rate  of  BR  decay  slows,  and  the  final  BR  is  stabilized  at  20% 
to  40%  of  the  initial  value.  During  extraction  in  PEM  plus  Triton,  the  cytoplasm  disperses  and  the  spindle 
appears  more  fibrous.  Spindle  BR  disappears  upon  cooling  (5°C  for  10  minutes)  two  minutes  after  lysis, 
but  not  upon  cooling  eight  minutes  later.  To  our  surprise,  when  either  Colcemid  (CLM,  10  or  20  nAf) 
or  colchicine  (100  ^A/)  was  included  at  lysis,  the  spindle  BR  did  not  decay  at  all  for  30  minutes.  Spindles 
were  stable  (BR  1 10-140%  of  initial  value)  in  lysis  medium  plus  CLM  with  or  without  detergent.  To  test 
whether  this  apparent  stabilization  was  due  to  CLM  itself  or  an  unknown  contaminant,  we  replaced 
CLM  in  the  lysis  buffer  with  its  inactive  analog  lumicolcemid  (LCLM),  prepared  by  irradiating  the  same 
CLM  solution  with  366  nm  light.  When  spindles  are  isolated  with  buffers  containing  LCLM  (20  nM), 
the  BR  decay  curve  closely  resembles  the  curve  for  spindles  isolated  in  PEM  buffer  alone.  Because  the 
LCLM  solution  should  contain  the  same  contaminants  as  the  CLM  solution,  it  appears  unlikely  that  the 
stabilization  observed  in  CLM  is  due  to  an  unknown  contaminant.  While  20  ^M  CLM  stabilizes  the 
isolated  spindle  for  over  30  minutes,  this  same  dose  of  CLM  induces  complete  loss  of  spindle  BR  in  8 
minutes  in  the  living  cell.  Therefore,  the  isolated  spindle  is  no  longer  CLM-labile,  it  is  stabilized  instantly 
by  exposure  to  PEM  buffer  containing  CLM,  and  it  loses  cold  lability  in  a  few  minutes. 

Supported  by  NSERCC  post-graduate  fellowship  (D.A.L.),  U.  of  PA  General  Honors  Program,  ACS 
PF-2130  (T.O.),  and  grants  NSF  PCM  79-22136  and  NIH  7R01-GM31617  (S.I.). 


Dissociation  constants  of  dimeric  actin  cross-linking  proteins.  GEORGE  Q.  DALEY 
AND  NORMA  ALLEWELL  (Wesleyan  University,  CT). 

A  number  of  structurally  similar  reversibly  associating  proteins  purified  from  cytoplasmic  extracts 
cross-link  actin  filaments  to  form  isotropic  gels.  The  cross-linking  efficiency  of  these  proteins  depends 
upon  not  only  their  affinity  for  actin  but  also  the  extent  to  which  they  self-associate.  Determination  of 
the  equilibrium  constants  for  self-association  and  actin-binding  permit  quantitative  comparisons  of  the 
ability  of  these  proteins  to  contribute  to  the  gelation  of  cytoplasmic  actin. 

Both  values  were  measured  for  the  dimeric  actin  cross-linking  protein  filamin.  Filamin  was  purified 
from  chicken  gizzard  by  the  method  of  Feramisco  and  Burridge  (1980,  /  Biol.  Chem.  255:  1 194-1 199). 
The  binding  affinity  (KD)  of  filamin  for  F-actin  was  determined  by  a  co-sedimentation  assay.  F-actin, 
(2.4  nAf),  was  mixed  with  various  concentrations  of  filamin  in  0.1  M  K.C1,  1  mA/Ca/EGTA  (R  =  0.05), 
1  mA/  Mg-ATP,  20  mM  PIPES,  pH  6.8  (volume  0.2  ml)  and  incubated  for  30  min  at  25°C.  F-actin  and 
filamin  alone  served  as  controls.  The  solutions  were  centrifuged  at  30  PSI  (100,000  X  g)  for  30  min  at 
RT  in  an  air-driven  microcentrifuge.  Virtually  all  of  the  actin  (90%)  but  none  of  the  filamin  (4%)  in  the 
controls  pelleted  under  these  conditions.  Filamin  which  sedimented  in  the  presence  of  F-actin  was  there- 
fore assumed  to  be  bound.  To  determine  the  quantities  of  actin  and  filamin  10%  SDS-polyacrylamide 
gels  of  the  pellets  and  supernatants  stained  with  coomassie  blue  R  were  scanned  densitometrically. 
Scatchard  analysis  of  the  binding  data  yielded  a  KD  of  2.1  X  10  7  M  and  a  binding  ratio  of  1  filamin 
dimer  for  every  13  actin  monomers. 

The  KD  for  the  monomer-dimer  equilibrium  was  determined  by  analytical  gel  chromatography 
(Valdes  &  Ackers  1978,  Meth.  En:.  61:  125-142)  using  high  pressure  liquid  chromatography  (HPLC 
TSK-400  column,  Biorad).  From  the  measured  KD  value  of  2  X  10  6  M  a  free  energy  of  association  for 
the  filamin  dimer  was  calculated  to  be  -7.7  kcal/mole. 

Supported  by  NIH  Training  Grant  GM-3 11 36-04.  HPLC  equipment  was  generously  loaned  by 
Biorad. 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Isolation  and  study  ofmetaphase  and  anaphase  meiotic  spindles  from  Chaetopterus 
oocytes.  DENNIS  GOODE  AND  VIDYA  SARMA  (University  of  Maryland, 
College  Park). 

We  have  developed  methods  to  isolate  and  study  the  meiotic  spindles  from  Chaetopterus  oocytes, 
which  enter  and  become  naturally  arrested  in  first  meiotic  metaphase  shortly  after  exposure  to  natural 
sea  water.  Since  oocytes  are  much  larger  than  the  spindles  and  contain  birefringent  yolk  granules,  a  useful 
first  step  is  to  prepare  "mini-cells."  Metaphase  eggs  resuspended  in  calcium-free  sea  water  are  placed 
over  a  1  M  sucrose  solution  containing  10%  calcium-free  sea  water  and  centrifuged  at  25°C  and  24,000 
<  g  for  10  min.  The  cells  removed  from  the  interface  between  the  solutions  are  primarily  small,  clear 
cells  containing  meiotic  spindles.  These  spindles  remain  arrested  in  metaphase  and  can  be  increased  or 
decreased  in  size  by  temperature  shifts  or  mitotic  inhibitors.  For  example,  exogenous  maytansine  (2.5 
nM)  produces  spindle  disassembly  in  3  min.  Anaphase  mini-cells  are  prepared  by  adding  sperm  1  min 
before  centrifuging  fertilized  eggs  on  a  sucrose  cushion  for  5  min.  When  mini-cells  are  lysed  with  Nonidet 
P-40  in  microtubule  assembly  buffer  plus  2  mg/ml  bovine  brain  tubulin  and  10~3  M  GTP,  the  birefrin- 
gence of  the  spindles  is  enhanced.  When  lysed  in  the  same  medium  containing  2  mg/ml  dichlorotria- 
zinylaminofluorescein-labeled  microtubule  proteins  and  incubated  for  12  min  at  32°C,  the  entire  spindle 
becomes  fluorescent;  but  when  incubated  for  5  min,  fluorescence  is  concentrated  in  two  regions  just 
poleward  from  the  metaphase  chromosomes.  Since  clean  spindles  are  difficult  to  isolate  from  oocytes, 
we  use  mini-cells  as  an  intermediate  step  in  isolation.  Mini-cells  are  washed  in  isolation  medium  without 
detergent,  then  pelleted  and  resuspended  in  100  times  their  volume  of  isolation  medium  (2  A/glycerol, 
50  mM PIPES,  5  mA/  EGTA,  0.5  mM  MgCl2,  and  0.5%  Nonidet  P-40  at  pH  6.8)  until  cells  lyse.  Anaphase 
meiotic  spindles  can  be  isolated  from  anaphase  mini-cells  for  analysis  and  comparison  with  metaphase 
spindles. 

Supported  by  NSF  grant  PCM-801 1474. 

Regions  of  microtubule  assembly  in  isolated  spindles  o/'Spisula  solidissima.  LEAH 
T.  HAIMO  (University  of  California,  Riverside)  AND  BRUCE  R.  TELZER. 

Microtubules  (MTs)  may  be  described  by  two  parameters,  their  structural  polarity  and  their  polarity 
of  assembly.  MTs  within  a  half  spindle  have  been  shown  previously  to  be  oriented  with  their  plus  ends 
located  at  the  equatorial  plate.  Studies  were  now  undertaken  to  determine  the  polarity  of  assembly  of 
these  MTs.  Spindles  were  isolated  from  oocytes  of  Spisula  solidissima  and  then  incubated  with  Tetra- 
hymena  or  Chlamydomonas  dynein  which  resulted  in  the  uniform  decoration  of  all  spindle  MTs.  These 
dynein-decorated  spindles  were  then  incubated  in  6S  tubulin  which  was  incorporated  into  the  spindles 
as  indicated  by  an  increase  in  their  birefringence  and  in  size  of  asters.  Gel  electrophoresis  revealed  that 
both  dynein  and  6S  tubulin  cosedimented  with  the  spindles.  Electron  microscopy  was  undertaken  to 
distinguish  between  dynein-decorated,  native  MTs  and  undecorated,  newly  assembled  MTs.  Undecorated 
MTs  were  observed  surrounding  the  periphery  of  the  spindle.  In  addition,  within  the  spindle  the  number 
and  length  of  undecorated  MTs  occurred  with  increasing  frequency  nearer  the  equator.  That  native 
spindle  MTs  did,  in  fact,  elongate  during  the  incubation  in  tubulin  was  suggested  by  the  observation  that 
some  MTs  possessed  two  domains,  one,  closer  to  the  pole,  dynein  decorated,  and  the  other,  distal  to  the 
pole,  undecorated.  These  domains  represent  native  and  newly  assembled  regions  of  MTs,  respectively. 
In  summary,  new  MT  assembly  occurred  primarily  around  the  periphery  and  in  the  equatorial  region 
of  isolated  spindles.  Experiments  are  currently  underway  to  determine  the  polarity  of  assembly  of  the 
kinetochore  MTs. 

Supported  by  grants  from  the  N.I.H.,  Univ.  of  Calif.  Committee  on  Research,  and  American 
Philosophical  Society  (L.T.H.)  and  by  grants  from  the  N.I.H.,  Pomona  College,  and  the  William  and 
Flora  Hewlett  Foundation  Grants  of  Research  Corporation. 

Further  studies  on  the  ultrastructure  and  distribution  of  lateral  line  and  ocular-as- 
sociated structures  (possibly  sensory)  in  a  marine  teleost  (Stenotomus  chrysops). 
CLIFFORD  V.  HARDING,  STANLEY  R.  SUSAN,  WOO-KUEN  Lo,  S.  GREGORY 
SMITH,  AND  VINAY  REDDY  (Kresge  Eye  Institute,  Wayne  State  University, 
Detroit,  MI). 

Epidermal  projections  near  the  eye  and  lateral  line  system  have  been  observed  in  the  scup.  Each 
projection  consists  of  four  cell  types:  a  single  core  cell,  a  monolayer  of  epithelium,  modified  epithelial 
cells  at  the  basal  region  (collar  cells)  where  the  projection  bends  passively  in  response  to  water  movements, 
and  the  bulb  cell,  which  has  a  complex  and  unique  structure.  The  distal  end  of  the  bulb  cell  has  a 


ACTIN,  MICROTUBULES,  ETC.  359 

prominent  bulb,  connected  by  a  long  cytoplasmic  stem  to  the  cell  body  located  in  the  epidermis  proper. 
The  stems  are  completely  enveloped  by  epithelial  cells,  each  of  which  envelops  a  portion  of  the  stem, 
and  shows  extensive  desmosomal  connections  where  one  portion  of  the  cell  meets  the  other  portion  of 
the  same  cell  (like  a  collar  button).  These  epidermal  cells  give  the  impression  of  primitive  Schwann  cells. 
The  bulb  cells  (which  number  6-8  and  run  parallel  to  the  core  cell)  are  also  unique  among  the  four  cell 
types  in  not  having  any  desmosomal  connections.  The  overall  arrangement  of  the  cells  within  the  pro- 
jections suggests  a  sensory  function,  perhaps  the  detection  of  the  direction  of  water  movement  (with  the 
bulb  cells  serving  as  differential  detectors  of  the  direction  of  projection  bending).  As  yet,  however,  we 
have  no  definitive  evidence  for  nerve  connections  with  any  portion  of  the  projection.  Studies  on  distri- 
bution show  that  the  number  of  projections  per  scale  is  several-fold  higher  dorsal  to  the  lateral  line  as 
compared  to  ventral  regions.  The  single  row  of  specialized  scales  along  the  lateral  line  form  a  distinct 
line  of  demarcation  between  the  dorsal  scales  with  large  numbers  of  projections  and  the  ventral  scales 
with  low  numbers.  Further  studies  of  distribution  may  provide  additional  clues  about  the  function  of 
these  small  but  complex  structures. 

Supported  by  NIH  grants  EYO-7034,  EYO-1874,  and  by  RPB,  Inc. 

Some  membrane  structural  changes  accompanying  morphogenetic  changes  in  Tet- 
rahymena.  LINDA  A.  HUFNAGEL  (University  of  Rhode  Island). 

The  surface  of  the  ciliated  protozoan  cell  is  covered  by  three  membrane  layers,  the  plasma  membrane 
(PM)  and  the  inner  and  outer  membranes  of  numerous  cortical  alveoli  which  directly  underlie  the  PM. 
How  these  membranes  are  assembled  is  not  understood  in  spite  of  an  intimate  role  in  cortical  morpho- 
genesis. Therefore,  freeze-fracture  EM  has  been  used  to  analyze  membrane  changes  accompanying  two 
developmental  sequences  in  the  ciliate  Tetrahymena  thermophila.  These  sequences  are  1)  tip  transfor- 
mation, a  remodeling  of  the  anterior  tip  of  the  cell  preceding  pairing  during  conjugation,  and  2)  rapid 
cell  growth  induced  by  refeeding  of  cells  starved  overnight.  During  tip  transformation,  new  linear  assem- 
blies of  intramembranous  particles  (IMPs)  develop  within  the  protoplasmic  face  (PF)  of  the  PM,  marking 
the  future  boundary  between  remodeled  regions  of  the  cell  cortex  and  remaining  unaltered  cortical 
regions.  Oriented  PF  arrays  normally  found  anterior  to  these  new  assemblies  then  disappear  and  the  PF 
in  this  region  becomes  relatively  devoid  of  IMPs.  Also  in  tip  transforming  cells,  rosettes  of  particles, 
hitherto  undescribed  in  Tetrahymena,  appear  on  the  PF  of  the  membranes  covering  anteriorly  situated 
cilia.  These  rosettes  lie  close  to  the  ciliary  plaques.  Their  random  positions  suggest  that  they  may  be  free 
to  move  laterally  within  the  membrane.  While  the  functions  of  these  arrays  are  unknown,  it  seems  likely 
that  linear  assemblies  are  important  in  shape  changes  that  precede  cell  pairing  while  ciliary  rosettes  are 
related  to  recognition  between  mating  types.  In  starved-refed  cells  sampled  about  4'/2  to  5'/2  h  following 
refeeding,  two  different  types  of  smooth  membrane  vesicles  were  associated  with  the  alveolar  membranes 
and  the  PM.  In  addition,  periodic  blebbing  of  the  membranes  was  observed  in  refed  cells  but  not  starved 
controls.  These  results  suggest  several  routes  for  introduction  of  constituents  into  cortical  membranes 
of  rapidly  growing  cells. 

Evidence  for  the  association  of  high  molecular  weight  proteins  (MAP  2)  with  a  subset 
of  microtubules  in  vitro.  GEORGE  M.  LANGFORD  (University  of  North  Carolina, 
Chapel  Hill)  AND  ADRIAN  C.  LAWRENCE. 

Recent  studies  have  shown  that  two  populations  of  microtubules  can  be  identified  in  intact  neurons 
by  immunofluorescence  microscopy.  We  designed  experiments  to  test  whether  two  populations  of  mi- 
crotubules can  be  reconstituted  in  vitro  from  purified  brain  microtubule  proteins  (MTPs).  Our  results 
show  that  two  populations  of  microtubules  can  be  identified  in  samples  of  microtubules  reconstituted 
in  vitro.  These  findings  support  the  hypothesis  that  purified  brain  MTPs  self-assemble  with  sufficient 
molecular  specificity  to  preserve  the  molecular  identity  of  the  subsets  of  microtubules  which  existed  in 
the  cells.  For  these  experiments,  we  purified  MTPs  from  the  brain  of  cattle,  shark,  and  squid.  When 
analyzed  by  SDS-PAGE,  the  bovine  brain  MTP  samples  contained  two  types  of  microtubule-associated 
proteins  (MAPs),  the  high  molecular  weight  (HMW)  and  tau  proteins.  Shark  and  squid  MTPs  contained 
two  types  of  MAPs,  but  the  HMW  MAPs  of  these  two  organisms  were  different  from  those  of  cattle. 
When  assembled,  all  three  types  of  MTPs  showed  two  populations  of  microtubules.  The  two  populations 
were  identified  by  negative  contrast  electron  microscopy.  One  type  of  microtubule,  representing  the 
majority  population,  appeared  as  smooth,  straight,  randomly  dispersed  filaments.  The  other  type  appeared 
as  wavy  filaments  that  were  always  cross-linked  into  large  tangled  bundles.  The  basis  of  cross-linking  was 
found  to  be  due  to  the  presence  of  lateral  projections  that  decorated  the  surface  of  these  microtubules. 
The  lateral  projections  fit  the  description  of  HMW  MAP  2.  The  smooth  and  decorated  microtubules  had 
similar  rates  of  depolymerization  upon  dilution.  We  hypothesize  that  the  two  types  of  microtubules  arise 


360  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

by  the  simultaneous  formation  of  two  kinds  of  nuclei,  one  kind  containing  tau  and  the  other  HMW 
MAPs.  The  nuclei  may  form  by  the  cooperative  interactions  between  a  given  class  of  MAPs  and  tubulin. 
The  ratio  of  the  two  kinds  of  nuclei  probably  depends  upon  the  ratio  of  the  two  kinds  of  MAPs.  The 
two  forms  of  microtubules  are  thought  to  represent  the  axonal  and  dendritic  populations  of  microtubules 
in  neurons. 

(A.C.  Lawrence  was  a  student  in  the  Rockefeller  Foundation  Program  in  Life  Sciences  for  High 
School  Students.) 

Zonulae  occludentes  and  transepithelial  permeability  in  the  ocular  lens  epithelium. 
Woo-KuEN  Lo  AND  CLIFFORD  V.  HARDING  (Kresge  Eye  Institute,  Wayne  State 
University,  Detroit,  MI). 

The  existence  of  zonulae  occludentes  (tight  junctions)  in  ocular  lens  epithelium  is  uncertain.  By 
utilizing  a  "double  mounting"  method  in  freeze-fracture  electron  microscopy,  we  have  demonstrated  the 
presence  of  zonulae  occludentes  structures  in  the  lens  epithelia  of  human  and  frog  (Rana  pipiens)  for 
the  first  time.  It  was  found  that  these  zonulae  occludentes  are  always  located  between  the  lateral  mem- 
branes of  epithelial  cells  in  close  proximity  to  the  apical  end  of  the  cells.  The  zonulae  occludentes  are 
characterized  by  a  number  of  continuous  anastomosing  grooves  or  strands  on  the  E-face  of  the  membrane. 

The  transepithelial  barrier  function  of  zonulae  occludentes  in  the  lens  epithelia  of  frog  and  sea  bass 
is  determined  by  a  "wash  out"  procedure,  in  comparison  with  the  conventional  "non-wash"  procedure 
for  the  protein  tracer  HRP  (horseradish  peroxidase).  In  the  "wash  out"  experiments,  frog  lenses  were 
washed  in  tissue  culture  medium  TC-199  (70%)  for  various  periods  of  time  (2,  7.5,  10,  15,  and  20 
minutes)  immediately  following  15  minutes  of  enzyme  incubation  within  the  eye  (1%  HRP  in  TC-199 
was  injected  into  the  anterior  chamber).  The  lenses  were  then  fixed  and  processed  for  cytochemistry.  We 
have  found  that  within  various  time  intervals  of  washing,  HRP  reaction  product  is  consistently  blocked 
at  the  location  of  membrane  fusions  (zonulae  occludentes),  as  seen  with  thin-section  transmission  electron 
microscopy.  This  corresponds  to  the  location  of  zonulae  occludentes  found  in  the  freeze-fracture  studies. 
By  applying  the  same  "wash  out"  procedure  to  the  sea  bass  lenses,  we  have  obtained  results  similar  to 
those  found  in  the  frog  lens.  Thus,  these  data  strongly  suggest  that  there  are  zonulae  occludentes  in  the 
lens  epithelia  of  human,  frog,  and  sea  bass,  and  that  these  structures  do  provide  a  barrier  function  for 
the  transepithelial  diffusion  of  HRP  (molecular  weight  40,000  daltons). 

Supported  by  NIH  grants  EYO-7034,  EYO-1874,  EYO-3247,  and  by  RPB,  Inc. 

L-leucine  transport  by  isolated  toadjish  hepatocytes.  ROGER  PERSELL  AND  AUDREY 
E.  V.  HASCHEMEYER  (Hunter  College). 

Hepatocytes  were  isolated  from  20  ±  1  °C-acclimated  toadfish  (350-400  g)  by  liver  perfusion  in  situ 
with  a  Ca++-free  buffered  balanced  salt  medium,  followed  by  a  medium  containing  1.7  mA/CaCl2,  100 
units/ml  collagenase  Type  IV  (Sigma),  and  physiological  concentrations  of  19  amino  acids  (excluding 
leucine).  Cells  were  collected  by  combing,  washed  twice  with  collagenase-free  medium,  and  resuspended 
at  0.1  g/ml  in  240  mA/  NaCl,  5  mM  KC1,  1.7  mA/  CaCl2,  0.4  mA/  KH2PO4,  0.3  mM  Na2HPO4,  4  mM 
Na  HCO3,  5.6  mA/  glucose,  10  mM  HEPES  (pH  7.8),  and  1%  bovine  serum  albumin.  Transport  ex- 
periments were  carried  out  in  Beckman  microfuge  tubes  by  addition  of  100  ^1  cell  suspension  to  100 
n\  medium  containing  0.1  juCi  L-'4C-leucine,  0.2  MCi3H-inulin  and  additions  as  indicated  below.  Reaction 
was  terminated  after  times  of  5  s  to  3  min  by  centrifugation,  removal  of  the  supernatant,  and  rinsing  of 
the  cell  pellet.  Intracellular  uptake  of  l4C-leucine  was  determined  after  correction  for  extracellular  ra- 
dioactivity by  use  of  3H  recovery.  Uptake  at  0. 1  mA/  external  leucine  concentration  followed  first-order 
kinetics  (k  =  1.1  ±  0.1  min  '  at  21°)  to  a  plateau  at  t  =  2  min,  corresponding  to  a  space  occupied  of 
1-2  n\  per  cell  pellet,  after  which  a  continuous  slow  linear  uptake  occurred.  Concentration  dependency 
of  the  early  time  course  corresponded  to  Michaelis-Menten  kinetics  with  Km  =  0.52  mA/and  Vmax  =  760 
pmole/^il  •  min.  The  principal  inhibiting  amino  acids  (tested  at  2  mA/)  were  isoleucine  and  phenylalanine; 
smaller  effects  werr  noted  with  valine  and  methionine. 

Supported  by     iational  Science  Foundation  grants  PCM  79-21091  and  DPP  80-21454. 

Uptake  and  utiliza,;  m  of  L-alanine  by  10  species  of  bivalve  molluscs.  ROBERT  D. 
PRUSCH,  SCOTT  M.  GALLAGER,  AND  ROGER  MANN  (Woods  Hole  Oceano- 
graphic  Institution). 

The  influx  of  dissolved  L-alanine  into  isolated  gill  and  mantle  tissue  of  adult  specimens  of  10  species 
of  bivalves  was  examined.  Isolated  tissue  was  incubated  for  2  h  in  10  ml  of  sea  water  containing  20  \iM 
L-alanine  (I4C)  while  the  time  course  of  L-alanine  depletion  from  the  medium  was  followed. 


ACTIN,  MICROTUBULES,  ETC.  361 

Influx  was  consistently  higher  in  isolated  gill  rather  than  isolated  mantle.  Influx  rates  for  isolated 
gill  were,  in  decreasing  order:  40,  27,  16,  11,  9,  8,  6,  4,  3,  2  nmol/mg  dry  wt/h  for  Lyrodus  pedicellatus, 
Argopecten  irradians,  Solemya  velum,  Modiolus  modiolus,  Crassostrea  virginica,  Mya  arenaria,  Bankia 
gouldi,  Mytilus  edulis,  Teredo  navalis,  and  Mercenaria  mercenaria,  respectively.  Additions  of  0.5  mM 
cyanide  terminated  influx  in  all  cases  indicating  that  flux  was  effected  by  active  transport.  Although  the 
three  species  of  shipworms  examined  (L.  pedicellatus,  B.  gouldi,  and  T.  navalis)  possess  symbiotic  ni- 
trogen-fixing bacteria  in  their  gills  (Waterbury  and  Galloway,  in  prep.)  a  consistently  higher  influx  of 
amino  acid  was  not  evident  in  the  shipworms  when  compared  with  the  other  species  examined.  Uptake 
of  L-alanine  by  isolated  gill  of  L.  pedicellatus  followed  Michaelis-Menten  kinetics  with  a  Vmax  of  0.086 
^mol/mg  dry  wt/h  and  a  Km  of  about  20  nM  within  a  range  of  ambient  L-alanine  concentrations  of  1- 
200  nM. 

Fractionation  of  adult  B.  gouldi  following  both  20  and  90  hour  incubation  periods  with  20  p.M  L- 
alanine  showed  distribution  of  the  I4C  label  throughout  the  protein,  carbohydrate,  and  lipid  fractions. 
Pediveliger  larvae  of  B.  gouldi  exhibited  a  VmM  of  0.015  ^mol/mg  dry  wt/h  and  a  Km  of  about  14  pM. 
After  a  one  hour  incubation  in  1  nM  L-alanine  about  60%  of  the  I4C  label  was  accounted  for  in  larval 
tissue  and  about  20%  was  collected  as  respired  I4CO2. 

L-alanine  accumulated  from  the  sea  water  is,  therefore,  actively  metabolized  by  both  larval  and 
adult  shipworms  and  may  account  for  a  major  portion  of  their  nitrogenous  nutrition. 

Supported  by  Office  of  Naval  Research  Contract  NOOO 1 4-79-C-007 1  NR  083-004. 

Rapid  rates  of  colchicine-  or  colcemid-induced  spindle  microtubule  disassembly  in 
vivo:  implications  for  the  mechanism  of  microtubule  assembly.  E.  D.  SALMON, 
M.  MCKEEL,  T.  S.  HAYS,  AND  C.  RIEDER  (Dept.  of  Biology,  University  of  North 
Carolina,  Chapel  Hill). 

Following  perfusion  of  metaphase  and  anaphase  cells  with  culture  media  containing  10  mM  col- 
cemid,  spindle  birefringent  retardation  (BR)  decreased  rapidly  to  10  percent  of  its  initial  value  within 
a  characteristic  period,  T,  of  30-60  sec  for  first  mitotic  spindles  of  Lytechinus  variegatus  (24°C),  first 
meiotic  spindles  of  Chaetopterus  pergamentaceus  (24°C),  and  for  Ptk,  tissue  culture  cells  (34°C).  BR 
was  measured  in  the  central  half-spindle  region  by  a  calibrated  voltage  from  a  video  "spot  meter." 
Elimination  of  plasma  membrane  permeability  effects  was  achieved  by  the  microinjection  of  colchicine 
or  colcemid  into  early  division  Lytechinus  cells.  For  intracellular  colchicine  concentrations  of  0. 1-5  mM, 
T  =  1 5-25  sec,  independent  of  concentration.  Below  0. 1  mM,  T  depended  inversely  on  the  concentrations 
of  colchicine  or  colcemid.  Ultrastructural  analysis  showed  that  the  rapid  loss  of  spindle  BR  was  due  to 
depolymerization  of  non-kinetochore  fiber  MTs;  kinetochore  fiber  MTs  were  differentially  stable.  Lum- 
icolchicine  at  0.5  mM  intracellular  concentrations  had  no  effect  on  spindle  BR  or  cell  division.  If  colchicine 
and  colcemid  block  only  the  assembly  reactions  by  binding  to  subunits  in  the  spindle  tubulin  pool,  then 
the  intrinsic  rate  of  dissociation  of  tubulin  from  non-kinetchore  MTs  is  600  dimers/sec  for  an  average 
initial  MT  length  of  7.5  ^m  and  T  =  20  sec.  This  rate  is  about  50-fold  greater  than  reported  rates  of 
dimer  association  to  a  single  end  of  a  MT  in  vitro  at  a  tubulin  critical  concentration  of  2  ^M.  Consequently, 
subunit  exchange  may  occur  at  multiple  sites  along  spindle  MTs  in  vivo. 

Supported  by  NIH  grant  24364. 

Interactions  of  several  heavy  metals  with  L-leucine  transport  in  the  intestine  of  the 
toadfish,  Opsanus  tau.  R.  Socci,  N.  CURTIS,  A.  FARMANFARMAIAN  (Rutgers 
University),  AND  A.  ZWEIFACH. 

It  is  known  that  heavy  metals  inhibit  enzymes  and  transport  systems  in  various  mammalian  tissues. 
We  have  examined  the  effect  of  HgCl2,  CH3HgCl,  CdCl2,  and  SrCl2  on  the  intestinal  absorption  of  0.25 
mM  L-leucine  by  the  toadfish,  Opsanus  tau. 

The  absorption  of  l4C-labeled  L-leucine  from  buffered  fish  Ringer's  in  vitro  was  measured  in  the 
presence  and  absence  of  a  heavy  metal.  3H-inulin  was  used  as  a  water  marker.  Hg2+  produced  significant 
(P  <  0.01)  inhibition  of  uptake  at  all  concentrations  tested — 20%  at  2.5  ppm,  40%  at  5  ppm,  54%  at  10 
ppm,  and  67%  at  20  ppm.  CH3Hg+  inhibited  uptake  significantly  (P  <  0.05)  by  22%  at  5  ppm,  30%  at 
10  ppm,  and  46%  at  20  ppm,  but  not  at  2.5  ppm.  Neither  Cd2+  nor  Sr2"^  produced  significant  inhibition 
in  the  time-concentration  range  tested  (2.5-20  ppm  at  10  min).  Preliminary  experiments  showed  that 
the  removal  of  Ca2+  or  Mg2+  from  the  Ringer's  caused  a  moderate  level  of  inhibition  in  leucine  transport, 
this  inhibition  was  not  synergistic  with  that  of  Hg2+  when  the  latter  was  added  at  10  ppm. 

In  part  supported  by  NOAA  Sea  Grant  No.  NA82AA-D-00065  and  the  Center  for  Coastal  and 
Environmental  Studies  and  Busch  Fellowship  from  the  Bureau  of  Biological  Research  to  one  of  us  (R.S.), 
Rutgers  University. 


362  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Vigorous  movement  of  sand  dollar  sperm  during  extraction  with  Triton  X-100.  CYN- 
THIA L.  SUNDELL  AND  TIM  OTTER  (Marine  Biological  Laboratory). 

Sand  dollar  (Echinarachnius  parmd)  sperm  treated  with  Triton  X-100  become  hyperactive  for  about 
ten  seconds,  then  stop  until  they  are  reactivated  by  addition  of  ATP.  We  studied  this  brief  transition 
("the  burst")  from  living  sperm  to  "Triton-model"  using  frame-by-frame  motion  analysis  of  video  re- 
cordings. Living  sperm  near  a  glass  surface  normally  swim  in  circular  or  open  spiral  paths  of  characteristic 
diameter.  Flagellar  movement  is  nearly  symmetric.  Occasionally,  the  sperm  stop  in  cane-shaped  config- 
urations for  one  or  two  seconds  before  resuming  normal  motility  (similar  to  behavior  described  by 
Gibbons  1980,  /.  Cell  Biol.  84:  1-12).  During  the  "burst"  induced  by  addition  of  detergent,  flagellar 
beating  is  highly  asymmetric.  Many  sperm  appear  C-shaped,  with  a  large  principle  bend  and  little  or  no 
compensating  reverse  bend.  Since  the  flagellum  is  curved  to  one  side,  the  sperm  swim  in  a  tight  spiral 
path.  Finally  they  thrash  about  in  a  frenzied  pitching  motion.  Neither  behavior  was  seen  in  untreated 
sperm.  At  the  end  of  the  burst,  the  sperm  stop  abruptly  in  configurations  resembling  "rigor  waves." 
Many  sperm  have  two  round  swellings  at  the  midpiece.  After  extraction  (with  0. 1 5  M  KC1,  2  mM  MgSO4, 
2  nuWTris-HCl,  5  mM  CaCl2,  1  mM  EDTA,  0.04%  Triton  X-100,  pH  8.2),  ATP-reactivated  sperm  beat 
with  nearly  symmetric  waveforms  and  swim  in  straight  lines.  Thus,  the  asymmetric  beating  seen  during 
the  burst  is  not  due  to  a  permanent  structural  change  in  the  flagellum  upon  extraction.  To  test  whether 
a  release  of  mitochondrial  ATP  causes  the  burst,  we  immobilized  sperm  with  DNP  ( 1  mM  in  filtered  sea 
water)  prior  to  extraction  and  reactivation.  Surprisingly,  DNP-immobilized  sperm  also  repond  with  a 
typical  burst.  Therefore,  treating  sperm  with  the  extraction  buffer  causes  a  burst  in  activity  that  is  not 
due  to  a  sudden  release  of  mitochondrial  ATP,  and  that  briefly  restores  motility  to  DNP-immobi- 
lized sperm. 

Support:  U  of  PA  General  Honors  Course  Program,  NIH  7RO 1  GM  3 1 6 1 7  and  NSF  PCM79-22 1 36 
(S.I.),  ACS  PF  2130  (T.O.),  and  PHS  5T32GM-07229-07  (C.S.). 

ECOLOGY 

Effects  of  eutrophication  on  the  increase  of  chlorophyll  a  in  phytoplankton  from 
coastal  waters.  OSIRIS  BOUTROS  (University  of  Pittsburgh  at  Bradford,  Bradford 
PA  16701),  NINA  CARACO,  WILLIAM  DENNISON,  AND  IVAN  VALIELA. 

Phytoplankton  nutrient  enrichment  experiments  were  performed  in  sea  water  (Vineyard  Sound, 
32ppt  salinity)  and  brackish  water  (Salt  Pond,  28  ppt  salinity;  Siders  Pond,  4  ppt  salinity),  all  from  the 
Falmouth,  MA  area. 

The  effects  of  enrichment  were  assessed  by  fluorometric  and  spectrophotometric  determination  of 
chlorophyll  a. 

Combined  additions  of  nitrogen  and  phosphorus  produced  the  greatest  increases  in  growth  in  all 
three  waters  with  the  greatest  increases  recorded  in  Salt  Pond  water.  Phosphorus  increased  growth  in 
Siders  Pond  water  almost  as  much  as  the  combined  addition  of  nitrogen  and  phosphate  but  had  little 
or  no  effect  in  Vineyard  Sound  and  Salt  Pond  waters.  Nitrogen  addition  produced  a  greater  response  in 
Vineyard  Sound  water  than  in  Salt  Pond  and  had  no  effect  on  growth  in  Siders  Pond. 

Phosphorus  was  limiting  in  the  low  salinity  water  (Siders  Pond),  and  nitrogen  was  limiting  in  higher 
salinity  waters  (Salt  Pond  and  Vineyard  Sound). 

Frequency  of  resistance  to  selected  antibiotics  and  heavy  metals  and  the  occurrence 
of  plasmids  in  enteric  bacteria  from  a  marine  source.  SUSAN  BOUTROS  (Uni- 
versity of  Pittsburgh  at  Bradford,  Bradford,  PA  16701),  WILLIAM  REZNIKOFF, 
JEFF  GARDNER,  AND  NANCY  V.  HAMLETT. 

A  previous  study  of  resistance  in  enteric  bacteria  from  two  marine  ponds  in  the  Woods  Hole  area 
found  high  levels  of  i  -slstance  to  kanamycin,  ampicillin.  and  mercury  (Javero  1981,  unpublished).  The 
present  study  was  done  to  determine  the  current  levels  of  resistant  bacteria  in  the  same  marine  ponds 
and  whether  these  bacteria  possess  plasmids. 

Bacteria  were  collected  from  water  samples  from  Eel  Pond,  Great  Pond,  and  Salt  Pond  by  filtration 
through  nitrocellulose  membranes  (0.2  ^m  pore  size).  Enteric  bacteria  were  selected  and  the  proportions 
of  salt-tolerant,  metal-resistant,  and  antibiotic-resistant  bacteria  determined  by  placing  the  filters  on 
MacConkey  agar,  MacConkey  agar  with  2%  NaCl  (Mac  2);  Mac  2  +  ampicillin  (100  Mg/ml);  Mac  2 
+  kanamycin  (20  Mg/ml);  Mac  2  +  tetracyclin  (15  Mg/ml);  Mac  2  +  CdCl  (100  Mg/ml);  Mac  2  +  PbCl2 
(500  Mg/ml);  and  Mac  2  +  HgCl2  (10  mA/). 

Approximately  10%  of  the  enteric  bacteria  were  resistant  to  antibiotics  and/or  metals.  Of  75  resistant 


ECOLOGY  363 

strains  selected  for  further  study,  55%  were  not  salt  dependent  (non-marine),  and  45%  were  salt  requiring; 
25%  were  resistant  to  one  antibiotic  or  metal  and  75%  were  resistant  to  two  or  more.  The  most  common 
pattern  combined  resistance  to  one  antibiotic  with  resistance  to  one  or  both  metals.  No  Hg  resistance 
was  found.  Resistance  to  two  antibiotics  was  found  only  in  non-marine  forms. 

Twenty-seven  resistant  strains  were  screened  for  plasmids  using  the  technique  of  Kado  and  Liu 
(1981,  J.  Bad.  145:  1365-1373).  Plasmids  were  detected  in  60%  of  these  strains.  Resistance  to  Cd  and 
kanamycin  was  not  consistently  associated  with  plasmids.  Resistance  to  tetracyclin  and  ampicillin  was 
apparently  associated  with  small  plasmids,  but  additional  work  is  needed  to  demonstrate  that  resistance 
is  carried  on  transmissible  plasmids. 

Support  for  this  work  from  the  following  sources  is  gratefully  acknowledged:  S.B.  was  supported  by 
a  Faculty  Development  Grant  from  the  University  of  Pittsburgh  at  Bradford:  N.V.H.  was  supported  by 
a  Faculty  Research  Grant  from  Towson  State  University;  and  grants  were  received  from  NASA  NAGW- 
306  and  the  Foundation  for  Microbiology  to  Marine  Biological  Laboratory,  Woods  Hole. 

Seasonal  variation  in  the  flux  of  algal  pigments  to  a  deep-water  site  in  the  Panama 
Basin.  JONATHAN  J.  COLE  (Ecosystems,  Marine  Biological  Laboratory),  SUSUMU 
HONJO,  AND  NINA  M.  CARACO. 

A  moored  array  of  time-series  sediment  traps  was  deployed  for  an  entire  year  at  a  station  in  the 
Panama  Basin  (3680  m  deep;  5°ITN,  Sl°56rW)  with  traps  set  at  890,  2590,  and  3560  m.  At  each  depth, 
a  six-membered  rosette  of  collection  chambers  rotated  beneath  a  large  cone  (1.5  m2  diameter)  such  that 
each  chamber  collected  the  sediment  which  fell  into  the  cone  during  a  60-day  exposure.  The  chamber 
was  automatically  sealed  and  poisoned  after  the  exposure. 

At  each  depth  the  fluxes  of  algal  pigments,  organic  carbon,  and  carbonate  varied  seasonally.  Although 
most  of  the  carbonate  and  much  of  the  organic  carbon  was  associated  with  the  sinking  of  coccolitho- 
phorids,  the  flux  of  algal  pigments  was  not.  At  3560  m  the  peak  flux  of  phaeopigment  occurred  in 
February-March;  the  peak  flux  of  carbonate  occurred  in  June-July. 

Averaged  over  the  year  there  was  no  difference  in  the  quantity  of  algal  pigments  which  arrived  at 
the  three  depths.  Although  the  lowest  ratio  of  organic  carbon:phaeopigment  occurred  at  890  m 
(40:l;mg:mg)  and  the  highest  ratios  at  3560  m  (285:1),  on  the  average  there  was  no  trend  with  depth. 
These  results  suggest  that  decomposition  in  the  water  column  below  890  m  is  slow.  The  quantity  of 
phaeopigment  arriving  at  the  sea  floor  in  the  Panama  Basin  (80-280  ng-m  2-day~')  is  roughly  50  to 
100  times  greater  than  the  amount  sinking  to  2000  m  at  the  Tongue  of  the  Ocean  and  10  to  70  times 
less  than  the  amount  sinking  at  the  Peru  Upwelling. 

The  effects  of  oil  contaminated  sediments  on  the  growth  ofeelgrass  (Zostera  marina 
L.).  J.  E.  COSTA  (Boston  University  Marine  Program,  Marine  Biological  Lab- 
oratory). 

Eelgrass  (Zostera  marina  L.)  is  widespread  and  locally  important  in  temperate  and  cold  water  coastal 
ecosystems,  but  little  research  has  been  done  on  how  hydrocarbon  contamination  of  sediments  affects 
eelgrass.  No.  2  fuel  oil  recently  impacted  Buzzards  Bay,  MA,  and  in  many  sites  2  mg  hydrocarbons/g 
sediment  wet  wt  persisted  for  several  years. 

Sediment  was  collected  at  an  unpolluted  intertidal  site  near  Naushon  Island,  MA,  sieved  to  remove 
macrofauna,  and  contaminated  with  two  No.  2  fuel  oils:  American  Petroleum  Institute  (API)  reference 
III,  and  Baytown,  Texas  Exxon  (BTE)  refinery  oil.  Before  and  after  the  experiments,  eelgrass  seedlings 
were  weighed,  and  the  rhizomes  and  leaves  measured.  A  hole  was  punched  in  the  leaf  sheath;  leaf 
production  was  measured  by  the  outgrowth  of  scars.  There  were  7  treatments:  0  and  3.0  mg  API  oil/g 
sediment  wet  wt,  and  0,  0.2,  1.0,  2.1,  6.2  mg  BTE  oil/g  sediment  wet  wt.  Seedlings  were  planted  in 
an  outdoor  raceway  system  12  days  after  sediments  were  oiled  and  immersed,  and  harvested  three 
weeks  later. 

Eelgrass  seedlings  responded  similarly  to  both  oils.  Biomass  and  leaf  production  (measured  as  % 
weight  change,  rhizome  elongation,  and  relative  leaf  production),  showed  a  linear  decrease  when  plotted 
against  log  of  oil  concentration.  At  0.2  mg  oil/g  sediment,  leaf  production  and  weight  increase  were  16% 
and  40%,  respectively,  below  the  control.  Above  1 .0  mg  oil/g  there  was  actual  weight  loss,  50%  less  leaf 
production,  and  inhibition  of  root  and  rhizome  growth.  Under  field  conditions  wave  action  could  uproot 
these  plants.  Above  2. 1  mg  oil/g,  rhizomes  often  deteriorated,  leaves  were  shed,  and  many  plants  senesced. 
In  the  API  oil  experiment  chlorophyll  a  concentration  decreased  60%  providing  an  indication  of  the 
physiological  effects  that  occurred.  These  results  show  that  contamination  of  sediments  with  oil  could 
have  dramatic  effects  on  the  abundance  and  distribution  of  eelgrass. 

Thanks  to  J.  Capuzzo,  B.  Dennison,  and  I.  Valiela  for  their  assistance  and  advice.  S.  K.  Alexander 
(Texas  A  &  M)  supplied  the  BTE  oil.  This  work  was  in  part  funded  by  Sigma  Xi. 


364  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Role  oj  daily  light  period  and  intensity  in  photosynthesis  and  production  q/'Zostera 
marina  L.  (eelgrass).  W.  C.  DENNISON  AND  R.  S.  ALBERTE  (The  University  of 
Chicago). 

Photosynthesis  and  growth  responses  ofZostera  marina  L.  (eelgrass)  are  important  in  the  adaptation 
of  eelgrass  to  the  nearshore  marine  environment.  The  influence  of  light  regime  on  photosynthesis  and 
production  of  Z.  marina  was  examined  with  in  situ  manipulations  of  daily  light  periods  and  intensities. 
Underwater  lamps  and  light  shading  screens  were  placed  at  shallow  (1.3  m)  and  deep  (5.5  m)  stations 
in  an  eelgrass  bed  adjacent  to  the  Fisheries  Jetty  in  Great  Harbor,  Woods  Hole,  MA.  Underwater  lamps 
(300  /iE-nT2-s~')  extended  the  daily  light  period  by  4  to  6  hours  and  shade  screens  (-60  to  -80%  of 
ambient  light)  shortened  the  daily  light  period  by  3  to  5  hours  for  30  days  (June,  1982). 

Leaf  production  rates  at  the  deep  (5.1  dry  g-  m'2  -day^1)  and  shallow  station  (3.6  dry  g-  irT2  -day"1) 
were  reduced  by  5 1  and  56%,  respectively,  in  the  shading  experiments.  Underwater  lamps  increased 
production  by  15%  at  the  deep  station  but  had  no  effect  at  the  shallow  station.  Photosynthesis  versus 
irradiance  (P  vs  I)  curves  for  eelgrass  indicate  light  compensation  at  10  juE  •  m  2  •  s  '  with  light  saturation 
at  50  nE  •  m  2  •  s  '.  P  vs  I  curves  were  similar  in  shallow  station  experiments  but  variable  in  deep  station 
experiments.  Photosynthetic  unit  (PSU-O2)  size  was  doubled  by  shading  at  both  stations.  PSU  density 
was  reduced  by  50%  at  the  deep  station  shade  but  unaffected  by  the  shallow  station  shade.  An  increase 
(92%)  in  leaf  chlorophyll  content  occurred  only  at  the  shallow  station  shade  with  no  changes  in  chlorophyll 
amounts  or  a/b  ratios  evident  in  other  manipulations. 

These  results  indicate  1 )  there  is  potential  for  molecular,  cellular,  and  whole  plant  level  adjustments 
by  Z.  marina  to  changes  in  light  regime,  2)  changes  in  daily  light  period  play  an  important  role  in  eelgrass 
production  whereas  the  photosynthetic  apparatus  is  affected  by  light  intensity,  and  3)  different  response 
mechanisms  operate  in  plants  growing  at  shallow  and  deep  areas  of  the  eelgrass  bed. 

Research  supported  by  NSF  Grant  PCM-78 10535. 

Preliminary  investigation  of  water  quality  and  animal  mortality  at  MBL 's  Depart- 
ment of  Marine  Resources.  JULIE  EARLY  (Marine  Biological  Laboratory)  AND 
JOHN  VALOIS. 

The  effect  of  seasonal  changes  in  the  MBL  seawater  system  on  marine  specimens  kept  in  holding 
tanks  is  not  known.  MBL  sea  water  has  never  been  monitored  for  its  chemical  or  physical  changes. 
Without  this  information,  the  Laboratory  for  Marine  Animal  Health,  under  the  direction  of  Dr.  Louis 
Leibovitz,  can  not  obtain  a  clear  picture  of  what  stresses  are  placed  on  animal  stocks  in  holding  tanks. 

An  initial  examination  of  mortality  and  water  quality  is  presented.  Mortality  and  morbidity  are 
monitored  daily  while  water  quality  tests  are  completed  biweekly.  Water  samples  are  collected  from  the 
intake  and  discharge  of  each  of  the  buildings  housing  Marine  Resource's  animals.  Samples  are  obtained 
from  tanks  demonstrating  high  mortality  rates,  as  well.  The  water  quality  parameters  tested  include:  pH, 
salinity,  temperature,  turbidity,  ammonia,  nitrite,  nitrate,  phosphate,  sulfide,  copper,  and  iron. 

Values  obtained  at  this  point  are  as  follows:  pH,  7.6-8.2;  salinity,  30-32%o;  ammonia,  0.1-0.5  mg/ 
1;  nitrite,  0-0.05  mg/1;  nitrate,  0-0.4  mg/1;  phosphate,  0.004-0.2  mg/1;  sulfide,  0-0.005  mg/1;  copper,  0- 
0.02  mg/1;  iron,  0  mg/1;  turbidity,  0  FTU. 

Arbacia  punctnlata.  Strongylocentrotus  droebachiensis,  Asterias  forbesi,  and  Opsanus  tan  had  high 
mortality  rates  in  the  winter  due  to  the  extremely  low  water  temperatures  and  bacterial  attack.  Carcinus 
maenas.  Cancer  borealis,  and  Pagurus  pollicaris  had  high  mortality  rates  in  the  winter  and  spring  due 
to  starvation  and  cannibalism.  Raja  erinacea  suffered  in  the  summer  from  circulatory  disorders  caused 
by  the  difference  of  the  gas  pressures  in  the  water  in  which  they  were  caught  from  that  into  which  they 
were  placed  at  MBL. 

Contributing  factors  to  mortality  which  influence  water  quality  are  the  size  and  shape  of  the  tank, 
the  rate  of  water  flow,  the  number  of  individuals  per  tank,  the  condition  of  the  animals  before  being 
placed  in  the  tank,  amount  of  food  added,  and  weather  conditions  (air  temperature,  %  cloud  cover,  and 
precipitation). 

For  the  future,  it  can  be  assumed  that  seasonal  changes  in  both  the  chemistry  and  microorganisms 
in  MBL  sea  water  will  be  known.  Some  of  these  changes  will  be  traced  to  mortality  in  specimens.  This 
information  will  be  useful  in  the  construction  of  new  seawater  systems,  and  give  good  evidence  as  to 
when  recirculating  water  might  be  most  beneficial. 

Nutrient  flux  and  g\  .//?  of  the  red  alga  Gracilaria  tikvahiae  McLachlan  (Rhodo- 
phyceae).  RODNEY  M.  FUJITA  (Boston  University  Marine  Program,  Marine 
Biological  Laboratory). 

Nutrient  concentration  and  water  flow  rate  determine  the  availability  of  nutrients  to  macroalgae. 
Increasing  flow  rate  simultaneously  breaks  down  diffusion  gradients  near  the  thallus  and  increases  the 


ECOLOGY  365 

rate  of  nutrient  delivery.  Thus,  in  the  absence  of  diffusion  gradients  and  under  nitrogen-limited  conditions, 
nitrogen  flux  (flow  rate  X  concentration)  should  control  growth  rate. 

Gracilaria  tikvahiae  was  grown  outdoors  in  14-1  flow-through  tanks.  The  cultures  were  vigorously 
aerated  to  minimize  diffusion  gradients.  Gracilaria  required  approximately  2  weeks  to  acclimate  to  the 
experimental  conditions.  At  each  of  3  flow  rates  (5,  10,  and  20  culture  volumes/day)  replicate  tanks  were 
enriched  to  3  different  NH3-N  concentrations  (0.05,  0.10,  and  0.20  mA/).  The  N:P  ratio  was  maintained 
at  10:1.  This  resulted  in  5  different  nitrogen  flux  treatments  (3.5,  7.0,  14.0,  28.0,  and  56.0  millimoles  N/ 
day).  Specific  growth  rate  (SGR)  was  monitored  as  blotted  wet  weight  increase.  SGR  increased  as  a  linear 
function  of  nitrogen  flux  (r  =  0.79;  SGR  =  0.46  (N-flux)  +  4.9).  The  slope  was  significantly  different 
from  zero  (F  =  22.3,  P  <  0.001).  SGR  did  not  depend  on  concentration  or  water  flow  rate  alone.  These 
results  suggest  that  concentration  and  flow  rate  must  be  considered  together  (i.e.,  as  N-flux)  in  order  to 
understand  nutrient  uptake  and  nitrogen-limited  growth  in  Gracilaria. 

The  support  of  the  National  Wildlife  Federation  and  Sigma  Xi,  The  Scientific  Research  Society,  as 
well  as  the  able  assistance  of  C.  Errera  and  S.  Nolan,  is  gratefully  acknowledged. 

The  global  circulation  and  distribution  of  DDT.  DORIA  R.  GORDON  (The  Ecosystems 
Center,  Marine  Biological  Laboratory). 

The  release  of  persistent  pollutants  continues  to  increase  despite  evidence  of  negative  effects  in 
humans  and  other  organisms.  Use  of  the  insecticide  DDT  is  currently  extensive  in  the  tropics.  This  work 
has  focussed  on  the  consequences  of  a  southward  shift  in  application  of  this  compound. 

A  review  of  investigations  reveals  that  all  organisms  and  segments  of  the  biosphere  studied,  including 
air,  water,  and  sediments,  contain  detectable  levels  of  DDT  residues.  The  troposphere  has  an  average 
background  concentration  of  about  0.1  parts  per  trillion.  Nonetheless,  longterm  studies  indicate  that 
concentrations  of  DDT  are  decreasing  in  northern  regions  and  are  either  stable  or  increasing  in  the  tropics 
and  Southern  Hemisphere. 

A  simple  model  has  been  developed  to  examine  the  global  circulation  of  DDT  residues.  The  model 
reproduced  the  trends  of  contamination  observed  in  nature.  Residence  times  of  DDT  are  years  to  decades. 
Such  persistence  ensures  high  levels  of  residues  in  the  global  system  and  the  continuation  of  longterm 
dispersal.  The  analysis  was  limited  because  countries  are  not  required  to  disclose  the  actual  amounts  of 
the  insecticide  that  are  produced  or  consumed.  Thus,  the  quantity  and  distribution  of  the  DDT  released 
to  the  global  environment  is  unknown. 

Continued  investigation  of  DDT's  fluxes  is  appropriate  to  determine  where  accumulation  and  effects 
will  occur.  Critical  gaps  in  knowledge  result  from  the  lack  of  sampling  in  the  Southern  Hemisphere  and 
absence  of  information  on  production  and  use.  Experience  with  the  model  suggests  that  fluxes  in  the 
tropics  are  more  rapid  than  in  the  temperate  zone,  reducing  the  hazard  involved.  Longterm  global 
monitoring  of  both  specific  species  and  abiotic  reservoirs  should  provide  indications  of  trends  and  increase 
understanding  of  the  implications  involved  with  the  release  of  similar  compounds,  such  as  PCBs. 

Work  was  supported  by  The  Ecosystems  Center,  M.B.L.  through  a  grant  from  the  Culpeper  Foun- 
dation and  other  Center  funds.  The  assistance  of  Dr.  G.  M.  Woodwell,  Dr.  B.  Moore,  and  Ms.  J.  Dungan 
is  gratefully  acknowledged. 

Density  effects  on  growth  and  survival  of "Salicornia  bigelovii  and  S.  europaea.  JEAN 
M.  HARTMAN  (Marine  Biological  Laboratory)  AND  MARLIES  ENGLER. 

Two  annual  glassworts,  Salicornia  bigelovii  and  S.  europaea  (Chenopodiaceae),  are  common  in 
Great  Sippewissett  Salt  Marsh,  MA.  These  plants  are  found  primarily  in  areas  of  reduced  grass  cover, 
where  the  density  and  size  of  the  plants  vary  greatly.  We  tested  the  effect  of  density  on  the  growth  and 
survival  in  areas  with  dense,  monotypic  stands.  For  each  species,  we  selected  five  0.25  m2  plots,  over  a 
10  cm  range  of  elevations,  which  were  divided  into  25  0.01  m2  quadrats.  The  density  within  each  quadrat 
was  reduced  to  a  level  between  2  and  200  seedlings  in  early  June.  This  design  allowed  us  to  test  for 
between  plot  effects  as  well  as  density  effects.  In  mid-July  and  mid-August,  we  counted  the  number  of 
plants  surviving  in  each  quadrat  and  weighed  subsamples  of  each  density  treatment. 

Growth,  measured  as  the  average  weight  per  individual,  showed  no  significant  density-dependent 
effect  in  either  species.  The  dry  weight  of  individual  plants  varied  greatly,  but  no  predictable  pattern 
emerged  among  individuals  from  different  density  treatments.  Survival  rate  of  S.  bigelovii  showed  a 
highly  significant  density-dependent  effect,  determined  using  a  linear  regression  model.  However,  the 
data  are  scattered  and  only  25%  of  the  variability  can  be  explained  by  this  factor.  5".  europaea  showed 
no  significant  density-dependent  growth  characteristics.  Analysis  of  each  set  of  five  experimental  plots 
showed  that  elevation  is  significantly  correlated  with  growth  and  survival.  Also,  we  noted  that  intense 
herbivory  was  common  in  the  S.  europaea  plots. 

We  conclude  that  the  size  and  survival  rate  of  these  species  is  more  strongly  determined  by  site 


366  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

effects  than  by  intraspecific  interactions.  Site  effects  include  environmental  characteristics  such  as  ele- 
vation and  density-independent  biotic  effects  such  as  herbivory. 

This  work  was  supported  by  NSF  grant  OCE76- 19278  to  H.  Caswell.  Marge  Taylor  assisted  in  data 
collection. 

Denitrifying  bacteria  in  the  Great  Sippewissett  Salt  Marsh:  their  numbers,  diversity, 
and  distribution.  M.  E.  HEIMBROOK  (Dept.  Biological  Sciences,  Univ.  Northern 
Colorado,  Greeley,  Colorado  80639)  AND  J.  S.  POINDEXTER. 

Denitrification  in  the  salt  marsh  represents  a  major  nitrogen  loss  from  this  ecosystem  (Valiela  and 
Teal  1979,  Nature  280:  652).  Sediments  from  the  creek  beds,  pans,  high  and  low  marsh  areas  covered 
with  Spartina  alterniflora,  and  microbial  mats  sampled  in  the  present  studies  yielded  denitrifying  organ- 
isms from  cores  taken  as  deep  as  90  cm.  Gram-negative,  aerobic  to  microaerophilic,  polarly  flagellated 
coccobacilli,  rods,  vibrios,  and  spirilla  were  isolated  as  denitrifiers  from  enrichment  cultures  in  LANA 
medium  (1%  KNO3,  0.1%  sodium  lactate,  0.1%  sodium  acetate,  0.02%  NH4C1,  0.05  mM  Na2HPO4,  0.05 
m.U  KH2PO4,  in  80%  sea  water  and  adjusted  to  pH  8.0).  Marsh  sediments  diluted  ten-fold  were 
incubated  in  semisolid  LANA  medium  in  serum-stoppered  tubes  with  0. 1  atmosphere  of  acetylene  in 
air.  Acetylene  blocks  the  reduction  of  N2O  to  N2  (Yoshinari  et  al.  1977,  Soil  Biol.  Biochem.  9:  177)  and 
caused  the  accumulation  of  N2O.  an  intermediate  in  denitrification,  to  reach  levels  detectable  by  gas 
chromatography.  Most  probable  number  (MPN)  estimates  of  denitrifiers  in  the  semisolid  LANA  shake 
cultures  ranged  from  50/g  wet  sediment  from  the  creek  beds,  to  1800/g  in  the  sediments  under  the 
cyanobacterial  mats.  Populations  of  heterotrophic  bacteria  able  to  grow  in  the  medium  under  these 
conditions  ranged  from  3400/g  in  the  wet  pan  sediment  to  120,000/g  in  the  creek  bed  sediments.  The 
denitrifiers  in  the  sediments  under  the  cyanobacterial  mat  represented  20%  of  the  bacteria  growing  on 
LANA  medium,  but  only  0.04%  of  the  heterotrophs  from  the  creek  bed  were  denitrifiers.  The  numbers 
implied  by  the  shake  tube  MPN  procedure  may  be  underestimated  by  two  orders  of  magnitude  because 
of  heat  sensitivity  of  the  aquatic  bacteria.  However,  the  N2O  detection  method  offers  promise  as  a  rapid 
and  sensitive  method  for  detecting  denitrifiers.  Additionally,  the  release  of  CO2  by  growing  organisms 
serves  as  a  positive  internal  standard  for  growth  and  for  the  gas  chromatographic  analysis  of  the  metabolic 
products. 

Parts  of  this  research  were  supported  by  the  Foundation  for  Microbiology  and  NASA  NAGW-306. 

Regeneration  and  maturation  in  two  sympatric  Capitella  (Polychaeta)  sibling  species. 
SUSAN  D.  HILL  (Michigan  State  University),  JUDITH  P.  GRASSLE,  AND  SUSAN 
W.  MILLS. 

The  rapidity  with  which  certain  sibling  species  in  the  polychaete  genus  Capitella  can  reproduce  and 
increase  their  population  densities  in  disturbed  marine  habitats  is  well  known.  Sampling  these  populations 
has  commonly  shown  that  a  high  proportion  of  Capitella  show  evidence  of  tail  regeneration.  Laboratory 
experiments  have  been  conducted  to  investigate  the  effects  of  regeneration  on  maturation  and  fecundity 
in  the  co-occurring  Capitella  species  I  and  II.  Regeneration  was  initiated  by  amputating  tails  in  anes- 
thetized worms  of  known  age  and  parentage.  Weekly  observations  were  made  on  the  reproductive  state 
and  number  of  regenerated  tail  segments  in  individual  worms. 

(1)  In  Capitella  species  I  juveniles  at  «25°C  tail  regeneration  and  sexual  maturation  proceeded 
simultaneously.  (2)  At  20°C,  Capitella  species  I  males  isolated  to  promote  switching  to  a  hermaphrodite 
mode  went  through  oogenesis  equally  rapidly  whether  they  were  regenerating  or  not.  (3)  At  «25°C, 
Capitella  species  I  females  allowed  to  reproduce  4-5  times  regenerated  tail  segments  at  the  same  rate  and 
to  the  same  degree  as  females  that  were  in  the  early  stages  of  first  oogenesis.  That  is,  in  Capitella  species 
I  the  process  of  regeneration  does  not  appreciably  affect  the  processes  of  maturation  and  oogenesis. 

(4)  In  Capitella  species  II  at  20°C,  regeneration  significantly  slowed  the  rate  of  sexual  maturation 
from  the  late  juvenile  stage  in  developing  males  and  females.  (5)  In  a  separate  experiment  regenerating 
Capitella  species  II  females  had  a  significantly  lower  fecundity  over  an  8-week  period  than  nonregenerating 
worms.  That  is,  in  Capitella  species  II  regeneration  appears  to  occur  at  the  expense  of  reproduction, 
delaying  maturation  and  reducing  fecundity. 

In  the  field  heavy  predation  produces  a  high  proportion  of  regenerating  worms.  We  hypothesize  that 
the  resultant  delayed  sexual  maturation  and  reduced  fecundity  in  Capitella  species  II,  but  not  in  Capitella 
species  I,  may  account  in  part  for  the  differential  success  of  these  two  species  in  colonizing  new  habitats. 

This  research  was  supported  in  part  by  an  M.B.L.  Steps  Toward  Independence  Fellowship  and  a 
grant  from  the  Lerner-Gray  Fund  for  Marine  Research  of  the  American  Museum  of  Natural  History  to 
S.  D.  Hill,  and  by  N.O.A.A. 


ECOLOGY  367 

Effect  of  nitrogen  in  litter  and  in  ambient  water  on  microbial  respiration  in  Spartina 
decomposing  in  laboratory  microcosms.  ANDREW  C.  MARINUCCI,  JOHN  E.  HOB- 
BIE,  TERESA  L.  CORLISS,  AND  JOHN  V.  K.  HELFRICH  (The  Ecosystems  Center, 
Marine  Biological  Laboratory). 

The  effects  of  nitrogen  in  ambient  water  and  in  litter  on  the  decomposition  of  Spartina  alterniflora 
were  tested  with  laboratory  percolators.  The  CO2  respired  by  microbes  in  these  systems  was  used  as  an 
indicator  of  decomposition.  The  systems  were  operated  for  133  days  at  22-24°C,  contained  10  g  of  air- 
dried  shredded  litter,  and  were  kept  moist  with  artificial  sea  water  (24%o)  at  0.28-0.32  ml/mm.  Outflow 
air  was  trapped  in  0.2  N  KOH  and  titrated  periodically  to  determine  CO2  production.  Details  of  operation 
have  been  previously  described  (Marinucci  1982,  Biol.  Bull.  163:  53-69).  The  inflow  water  contained 
0,  10,  or  100  mg/1  N  as  NH4C1.  The  litter  was  collected  from  areas  at  a  salt  marsh  that  had  been  fertilized 
(Valiella  et  al.  1975,  /.  Appl.  Ecol.  12:  973-982);  as  a  result,  the  litter  in  the  experiments  contained  0.86, 
0.95,  1.22  and  1.33  %N/g  AFDW. 

The  results  of  this  experiment  in  which  effects  of  four  levels  of  internal  N  were  measured  at  three 
levels  of  N  in  the  surrounding  water  demonstrated  that  increasing  the  litter  N  caused  a  direct  increase 
in  total  CO2  production.  The  differences  among  CO2  produced  from  the  various  litter  types  resulted 
mainly  from  differences  in  rate  of  production  in  the  first  60  days.  The  CO2  production  rate  was  the  same 
for  all  litter  types  after  day  60.  Ambient  water  N  had  a  smaller  impact  on  total  CO2  production.  Maximum 
total  CO2  production  occurred  in  litter  incubated  with  10  mg  N/l.  However,  this  maximum  production 
resulted  from  a  very  high  rate  in  the  first  35  days.  After  day  60,  litter  receiving  0  mg  N/l  water  had  the 
highest  rate  of  CO2  evolution  with  10  mg  N/l  next  lower  and  100  mg  N/l  the  lowest.  Overall  conclusions 
were  that  litter  N  rather  than  water  N  had  the  greater  impact  on  decomposition. 

This  work  was  supported  by  NSF  DEB  79-05127. 

Potential  nitrification  rates  in  a  salt  marsh.  ERICH  R.  MARZOLF  (Colorado  College, 
Colorado  Springs,  CO  80903) 

Nitrification  is  an  important  process  in  the  cycling  of  nitrogen  in  coastal  marine  sediments.  Potential 
nitrification  rates  were  measured  in  sediments  from  Great  Sippewissett  Marsh,  Falmouth,  Massachusetts 
in  August,  1982.  Sediments  were  suspended  in  ammonia-enriched,  aerated  sea  water  and  shaken.  Tem- 
perature was  maintained  at  23-27°C.  Nitrification  was  measured  through  the  formation  of  nitrate  and 
its  subsequent  reduction  to  nitrite.  Rates  were  highest  in  the  top  2cm  and  decreased  exponentially  with 
depth.  Nitrification  rates  were  calculated  for  sediments  from  various  habitats  within  the  marsh.  Creek 
bank  sediments  had  the  highest  rate  (260  mg  N-NO3  •  hT1  •  m  2),  while  sediments  from  sandy  and  muddy 
creek  bottoms  had  rates  of  16.6  and  9.0  mg  N-NOj-rT1  -m~2,  respectively.  A  total  nitrification  rate  of 
26  kg  N-NOj  •  h  '  was  calculated  for  the  marsh  based  on  the  surface  area  of  each  habitat.  This  total  rate 
is  sufficient  to  account  for  the  estimated  difference  between  nitrate  import  and  combined  nitrogen  gas 
and  nitrate  export  (4740  kg  N-NOj)  in  Great  Sippewissett  Marsh. 


effect  of  habitat  structure  on  the  predator-prey  relationship  between  the  green 
crab,  Carcinus  maenas,  and  the  blue  mussel,  Mytilus  edulis.  EUGENE  C.  REVELAS 
(Marine  Sciences  Research  Center,  SUNY — Stony  Brook). 

Blue  mussels,  Mytilus  edulis,  abound  in  Nauset  Harbor,  Orleans,  MA  (Cape  Cod),  both  exposed 
on  the  mudflats  and  concealed  within  a  bordering  Spartina  alterniflora/ Fucus  vesiculosus  marsh.  The 
green  crab,  Carcinus  maenas,  a  voracious  predator,  is  abundant  on  these  mussel  beds.  The  effect  of  the 
marsh  habitat  on  this  predator-prey  relationship  was  investigated  in  seawater  tables  in  the  laboratory. 
Carcinus  of  various  sizes  were  starved  for  24  hours  and  then  allowed  to  forage  individually  on  equal 
numbers  of  different  sized  mussels  in  both  aquaria  devoid  of  vegetation  (representing  the  mudflat  habitat) 
and  in  a  simulated  "marsh."  The  "marsh"  was  constructed  by  sticking  plastic  straws  (Spartina)  into  holes 
in  a  piece  of  plywood  and  securing  Fucus,  collected  in  the  field,  around  the  straws.  During  each  foraging 
experiment  mussels  eaten  were  replaced  to  maintain  constant  prey  density. 

Carcinus  (3-5  cm  in  carapace  width)  were  found  to  predominate  in  the  field  based  on  three  '/2-hour 
searches.  In  the  laboratory,  Carcinus  (3-5  cm)  consumed  mussels  0-3  cm  in  length.  The  predation  rate 
(number  of  mussels  eaten  •  crab~'  •  day~')  in  the  "marsh"  was  70%  lower  than  in  the  "mudflat,"  apparently 
as  a  result  of  reduced  predator-prey  encounters.  The  size-frequency  distributions  of  the  marsh  and  mudflat 
Mytilus  populations  in  Nauset  Harbor  (estimated  by  measuring  all  mussels  within  randomly  tossed  10 
x  10  cm  quadrats)  are  explained  by  these  laboratory  results.  Mussels  0-3  cm  in  length  are  significantly 
more  abundant  in  the  marsh  (N  =  132)  than  on  the  mudflat  (N  =  35).  Also,  both  populations  show 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

enhanced  numbers  of  individuals  beginning  at  3  cm.  This  Mylilus  population  has  refuges  from  Carcinus 
predation  both  in  space  and  in  size. 

The  agreement  between  the  laboratory  results  and  the  field  observations  suggests  that  these  laboratory 
predator-prey  manipulations  reflect  natural  interactions.  These  data  indicate  that  natural  structural  com- 
plexities drastically  alter  the  predator-prey  relationship  between  Carcinus  and  Mytilus. 

The  effects  of  sulfide  on  cyanobacterial  photosynthesis  in  marine  microbial  mats. 
THOMAS  M.  SCHMIDT,  (Ohio  State  University)  AND  RICHARD  W.  CASTENHOLZ. 

The  cyanobacterial  or  blue-green  algal  mats  of  the  Little  Sippewissett  Marsh  in  Falmouth,  Massa- 
chusetts are  dominated  by  two  cyanobacteria,  Microcoleus  chthonoplastes  and  Lyngbya  aestuarii.  The 
concentration  of  hydrogen  sulfide  in  these  mats  reaches  at  least  1  mA/  during  the  summer  morning 
hours.  This  study  examines  the  effects  of  sulfide  on  the  photosynthetic  apparatus  of  these  cyanobacteria. 

Cores  of  the  mat  were  pre-incubated  in  the  light  for  3  hours  in  the  presence  or  absence  of  1  mA/ 
sodium  sulfide,  washed,  and  then  assayed  for  their  ability  to  photoassimilate  HI4CO3  in  the  presence  and 
absence  of  sulfide.  Field  samples  not  pre-incubated  with  sulfide  showed  a  50%  inhibition  of  H14CO3 
fixation  at  1  mA/  sulfide.  This  increased  to  70%  inhibition  at  2  mA/  sulfide.  HI4CO3  fixation  in  field 
samples  pre-incubated  with  sulfide  was  inhibited  by  60%  at  1  mA/  sulfide  and  75%  at  2  mA/  sulfide.  The 
addition  of  DCMU  (3-(3,4-dichlorophenyl)-l,l-dimethylurea)  to  field  samples  pre-incubated  with  or 
without  sulfide,  inhibited  HI4CO3  fixation  by  90%.  This  inhibition  was  not  decreased  by  the  addition  of 
1  mA/  sulfide  as  would  be  expected  if  sulfide-dependent  photosynthesis  were  taking  place. 

Pure  cultures  of  Lyngbya  aestuarii  were  pre-incubated  in  the  same  manner  as  the  field  samples. 
H14CO3  fixation  in  cultures  previously  unexposed  to  sulfide  was  inhibited  by  only  3%  under  1  mA/  sulfide. 
When  the  culture  was  pre-incubated  with  sulfide,  HI4CO3  fixation  was  inhibited  by  10%  when  again 
exposed  to  1  mA/  sulfide.  Two  mA/  sulfide  inhibited  H'4CO3  fixation  by  50%  regardless  of  previous 
exposure.  DCMU  again  inhibited  HI4CO3  fixation  by  90%  in  the  presence  or  absence  of  sulfide. 

Neither  the  field  samples  nor  the  Lyngbya  culture  showed  any  evidence  of  anoxygenic  photosynthesis, 
and  the  field  samples  showed  a  low  tolerance  to  sulfide.  These  results  are  somewhat  unexpected  since 
these  mat  organisms  are  subjected  to  frequent  exposures  to  sulfide  and  might  be  expected  to  have  retained 
or  evolved  a  greater  degree  of  sulfide  tolerance  or  sulfide-dependent  anoxygenic  photosynthesis. 

Wrack  accumulation  and  vegetation  structure  in  Great  Sippewissett  Salt  Marsh. 
EDWIN  K.  SILVERMAN  AND  JEAN  M.  HARTMAN  (Marine  Biological  Laboratory). 

A  descriptive  study  was  conducted  in  central  Great  Sippewissett  Salt  Marsh  to  compare  the  distri- 
butions and  associated  vegetation  of  wrack  (dead  plant  material  washed  into  the  marsh  by  tides)  and 
pannes  (algae-covered  or  bare  areas).  This  study  is  part  of  a  larger  project  being  conducted  by  one  of  us 
(J.  Hartman),  in  which  the  main  hypothesis  is  that  wrack  accumulation  can  cause  panne  formation. 

Percent  cover  measurements  inside  a  0.04  m2  quadrat  were  made  every  meter  along  twenty-two  line 
transects.  The  transects  were  placed  at  ten  meter  intervals  from  the  ocean  to  the  marsh  edge.  Percent 
cover  was  sampled  for  1781  quadrats.  Tidal  inundation  was  measured  on  325  of  the  sample  quadrats. 
Wrack  covered  8.0%  of  the  sample  quadrats. 

The  relations  of  wrack  and  panne  distribution  were  compared  to  tidal  inundation  and  distance  from 
the  ocean.  Most  of  the  wrack,  85.9%,  was  found  in  a  narrow  band  of  tidal  inundation  levels  between  13 
and  33  cm.  Most  of  the  pannes,  74.3%,  were  found  between  28  and  48  cm;  71.5%  of  the  pannes  were 
found  outside  the  main  band  of  wrack  accumulation.  A  similar  pattern  was  observed  with  distance  from 
the  ocean;  78.1%  of  pannes  were  located  beyond  the  range  of  most  of  this  year's  wrack  accumulation. 
Therefore,  if  wrack  causes  most  pannes,  its  long-term  distribution  differs  considerably  from  this  year's 
accumulation. 

The  relation  of  wrack  and  pannes  to  vegetation  structure  was  analyzed  using  R-type  principal 
components  analysis.  Wrack  quadrats  did  not  correlate  significantly  with  any  principal  component.  A 
principal  component  consisting  of  increased  short  Spartina  alterm (flora.  Salicornia  europea,  and  Salicornia 
biglovii,  and  decreased  large  S.  alterniflora  correlated  significantly  with  pannes  for  some  of  the  sample 
transects,  suggest  i. .?  -\  generalized  vegetation  type  for  pannes. 

This  work  was  supported  in  part  by  NSF  Grant  OCE  76-19278  to  Hal  Caswell.  E.K.S.  was  supported 
by  a  W.H.O.I.  Sumn,  Student  Fellowship. 

Role  of  shoot  photosynthesis  in  root-rhizome  respiration  in  Zostera  marina  L.  (eel- 
grass).  ROBERT  D.  SMITH,  WILLIAM  C.  DENNISON,  AND  RANDALL  S.  ALBERTE 
(The  University  of  Chicago). 

The  majority  of  productive  seagrasses  grow  in  anoxic  sediments  in  coastal  waters.  Because  the  nature 
of  the  respiratory  behavior  of  the  underground  tissues  in  these  species  is  essentially  unknown,  we  examined 


ECOLOGY  369 

the  role  of  shoot  photosynthesis  in  supporting  aerobic  respiration  in  the  root-rhizome  system  of  the 
temperate  seagrass  Zostera  marina  L.  (eelgrass). 

Uptake  and  release  of  oxygen  from  the  root-rhizome  system  of  Zostera  was  measured  polargraph- 
ically  in  a  two-chambered  apparatus  fitted  with  an  oxygen  electrode.  Root-rhizome  respiration  rates 
averaged  15.8  (±0.3)  nMol  O2  •  h  '  •  mg  '  (dry  wt).  Upon  illumination  of  the  shoot,  oxygen  transport  to 
the  root-rhizome  system  began  within  1 5  to  30  min  and  gave  oxygen  uptake  and  release  rates  of  -6.2 
to  +7.4  nMol  O2-rT'  -mg~'  (root-rhizome  dry  wt)  respectively,  or  -1.92  to  +0.46  nMol  O2-h~1-mg~1 
(shoot  dry  wt).  Within  10  to  20  min  after  the  shoots  were  placed  in  the  dark,  oxygen  transport  to  the 
root-rhizome  ceased.  Rates  of  oxygen  transport  to  the  root-rhizome  during  shoot  photosynthesis  ranged 
from  0.27  to  0.64  nMol  O2-rr'  -mg~'  (shoot  dry  wt).  These  results  show  that  (1)  shoot  photosynthesis 
is  responsible  for  oxygen  transport  to  the  root-rhizome  system;  (2)  oxygen  transport  to  the  root-rhizome 
is  rapidly  initiated  and  terminated  with  changes  in  the  shoot  light  regime;  (3)  shoot  dry  weight  is  highly 
correlated  to  the  rate  of  oxygen  transport  to  the  root-rhizome;  and  (4)  the  shoot:root-rhizome  ratio  is 
highly  correlated  to  the  oxygen  exchange  rate  with  the  sediments. 

We  have  demonstrated  that  shoot  biomass  controls  the  supply  of  oxygen  to  the  root-rhizome  system 
of  Zostera.  This  may  explain  the  observed  greater  shoot  biomass  at  depth  under  light-limited  conditions. 
In  addition,  since  daily  light  period  and  intensity  for  photosynthesis  decreases  with  depth,  the  availability 
of  oxygen  for  aerobic  root-rhizome  respiration  also  decreases  along  a  depth  gradient.  Consequently,  the 
period  of  root-rhizome  anaerobiosis  may  influence  the  depth  distribution  of  this  species. 

Semihmar  spawning  cycle  in  a  Woods  Hole  population  o/^Fundulus  heteroclitus. 
JEFFREY  J.  STODDARD  (Dept.  of  Zoology,  University  of  Wisconsin,  Madison, 
WI  53703). 

A  spawning  pattern  synchronous  with  the  semilunar  tidal  cycle  has  been  demonstrated  for  a  Mas- 
sachusetts population  of  Fundidiis  heteroclitus,  the  common  salt  marsh  killifish. 

Samples  were  collected  from  26  June  to  9  August  1982  from  the  Great  Sippewissett  Marsh,  Cape 
Cod.  Measurements  of  gonadosomatic  index  [(wet  gonad  weight/body  weight)  X  100],  percentage  of 
oocytes  in  the  final  maturational  stages,  and  mean  number  of  mature  oocytes  per  body  weight  showed 
clear  increases  immediately  before  each  new  and  full  moon.  Collections  of  young-of-the-year  fish  showed 
discrete  cohorts  consistent  with  semilunar  periodicity  in  spawning. 

The  spawning  rhythm  is  of  adaptive  significance  because  it  makes  possible  deposition  of  eggs  within 
the  high  creekbank  zone  thus  reducing  their  exposure  to  predators  and  strong  currents. 

Selection  for  moderately  halophilic  bacteria  by  gradual  salinity  increases.  A.  VEN- 
TOSA  (Dept.  Microbiologia,  Facultad  de  Farmacia,  U.  de  Sevilla,  Sevilla,  Spain), 

J.  S.  POINDEXTER,  AND  W.  S.  REZNIKOFF. 

Halophilic  bacteria  grow  optimally  in  media  containing  10%  sea  salts  to  saturation  (>40%)  and  are 
usually  isolated  from  hypersaline  habitats.  Because  naturally  occurring  hypersaline  sites  are  geographically 
discontinuous  and  generally  result  from  concentration  of  sea  water  by  solar  evaporation,  these  studies 
addressed  the  question  of  whether  oceanic  water  contained  halophilic  bacteria  and  so  could  serve  as  their 
medium  of  dispersal  between  hypersaline  sites.  One  previous  such  study  ( Rodriguez- Valera  el  al.  1979, 
Appl.  Env.  Microbiol.  38:  164)  reported  the  isolation  of  halophilic  bacteria  from  ocean  waters  off  the 
coast  of  Spain  within  15  km  of  onshore  salterns.  In  this  study,  bacteria  were  collected  by  filtration  from 
water  of  Vineyard  Sound,  whose  shores  lack  extensive  hypersaline  sites.  Two  samples  of  25  1  each  were 
filtered,  and  the  bacteria  from  each  sample  were  used  to  inoculate  a  medium  containing  a  complex  of 
organic  nutrients.  One  sample  was  incubated  initially  with  3%  sea  salts  and  periodically  received  fresh 
medium  containing  salts  adjusted  to  provide  salts  increments  of  3%.  The  second  sample  was  initiated 
at  10%  sea  salts,  and  salinity  was  increased  in  increments  of  4%.  Moderately  halophilic  bacteria,  which 
accounted  for  fewer  than  j^oo  of  the  viable  bacteria  present  in  the  sample  populations,  were  strongly 
favored  in  each  culture  when  salinity  reached  14-15%;  they  accounted  for  a  majority  of  each  population 
by  the  time  the  salts  concentration  reached  20%.  Non-halophilic  bacteria  (both  salt-tolerant  and  salt- 
dependent)  decreased  in  numbers  and  diversity  when  salts  concentrations  reached  14-15%;  at  higher 
salinities,  screening  of  clones  of  putative  "marine  bacteria"  revealed  them  to  be  moderate  halophiles  able 
to  grow  over  a  wide  (3-20%)  salinity  range.  The  quantitative  changes  in  both  cultures,  which  proceeded 
to  higher  salinities  on  different  schedules,  revealed  that  salinity  alone  accounted  for  enrichment  of  mod- 
erate halophiles.  The  results  of  these  laboratory  studies  imply  that  halophilic  bacteria  of  natural  hyper- 
saline sites  are  derived  from  sea  water,  and  are  enriched  during  the  course  of  solar  evaporation.  Further, 
they  predict  that  moderate  halophiles  become  predominant  in  such  sites  by  the  time  80%  of  the  water 
has  evaporated  and  salts  are  at  approximately  one-third  saturation.  A  collection  of  moderately  halophilic 
bacteria  has  been  accumulated  for  characterization. 

Parts  of  this  research  were  supported  by  the  Foundation  for  Microbiology,  and  NASA  NAGW-306. 


170  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

TTz^  development  and  geomorphology  of  Great  Sippewissett  Marsh  (Falmouth,  MA): 
the  Redjield  model  revisited.  MARC  WEISSBURG,  ALLYSON  SENIE,  GEORGE 
KOWALLIS,  AND  JOSEF  TREGGOR  (Dept.  of  Biology,  Central  Connecticut  State 
College,  New  Britain,  CT  06050) 

The  important  ecological  role  of  salt  marshes  has  resulted  in  their  being  the  focus  of  intensive 
scientific  study,  little  of  which  has  been  concerned  with  marsh  ontogeny.  The  notable  exception  is  the 
elegant  study  of  the  Barnstable  (MA)  Marsh  by  Arthur  Redfield  (1965,  Science  147:  50-55).  Utilizing 
data  from  sub-surface  soundings  and  analysis  of  water  content,  organic  content,  and  floral  dominance 
in  core  samples  we  have  developed  a  model  of  the  Great  Sippewissett  Marsh. 

Soundings  at  670  points  along  three  major  transects  revealed  peat  depths  of  23-653  cm.  Large  scale 
depressions  occurred  along  the  upland  margins  while  oscillations  in  topography  and  depth  of  peat  de- 
creased towards  the  sea.  Coring  sites  were  selected  with  reference  to  sub-surface  topography  and  26  cores 
were  exuded.  Based  upon  the  percent  of  water  and  organic  content  (OC),  sediment  horizons  were  char- 
acterized as  high  marsh  (60-90%  H2O;  35-69%  OC),  intertidal  (30-60%  H2O;  2-25%  OC),  or  fresh/ 
brackish  (85-92%  H2O;  70-90%  OC).  Visual  inspection  of  the  roots  and  rhizomes  provided  additional 
criteria  for  identification.  Saltmarsh  peat  extended  to  depths  of  400  cm  and  fresh/brackish  peat  to 
650  cm. 

Based  on  the  depth  of  saltmarsh  peat,  the  age  was  approximately  2900  years  and  generally  followed 
the  Redfield  model  of  development.  Certain  differences  were  evident:  Great  Sippewissett  developed 
through  the  establishment  of  saltmarsh  islands  which  gradually  coalesced  into  a  contiguous  high  marsh 
plain,  while  Barnstable  Marsh  expanded  from  fringe  areas.  The  colonization  of  upland  areas  occurred 
relatively  later  in  the  development  of  Great  Sippewissett  than  of  Barnstable. 

Factors  responsible  for  these  differences  were  variation  of  surface  topography  and  the  development 
of  fresh/brackish  water  wetlands  over  a  substantial  part  of  the  abutting  uplands  prior  to  the  commence- 
ment of  saltmarsh  development  at  Great  Sippewissett  Marsh. 

It  is  clear  that  localized  events  play  a  major  role  in  saltmarsh  ontogeny  and  must  be  considered  in 
any  model  of  saltmarsh  development. 

Germination  properties  of  a  marine  spore-forming  bacterium.  P.  WIER  (Dept.  EPO 
Biology,  Univ.  of  Colorado,  Boulder),  A.  KEYNAN,  AND  H.  O.  HALVORSON. 

Pigmented  aerobic  spore-forming  bacteria  were  isolated  from  the  Sippewissett  Marsh  (Singer  and 
Leadbetter  1974,  Biol.  Bull.  147:  499)  and  from  the  marshes  of  the  German  North  Sea  (Fahmy  1978, 
Ph.D.  Thesis,  Gottingen).  While  there  is  evidence  concerning  the  marine  nature  of  these  organisms,  their 
germination  properties  are  unknown.  The  germination  properties  of  one  such  isolate  (Hamlett,  1981) 
was  investigated.  This  strain  sporulates  well  on  Zobells  medium  and  produces  a  carotenoid  pigment  with 
maximum  absorption  at  492  nm.  Spores  were  purified  following  lysis  of  the  vegetative  cells  in  distilled 
water  and  density  centrifugation  through  50  to  60%  renografin.  Optimal  germination  required  a  short 
heat  activation  ( 10  min  at  60°C),  pH  7.8,  and  0.3  mM  adenosine,  85  mM  Na+,  and  about  70  mM  NH4+. 
No  germination  occurs  with  either  Na+  or  NH4+  alone.  NH4+  could  partly  replace  Na+  but  was  much 
less  effective  (Km  NH4+  =  2 1 4  rruV/;  Km  Na+  =  14.7  mM).  The  divalent  cation  and  dipicolinic  acid  (DPA) 
contents  of  3A10  and  two  related  marine  spore-forming  isolates  were  similar  to  those  of  terrestrial  strains 
(DPA,  7.5-12.1%;  Ca++,  2.1-2.7%;  Mg++,  0.17-0.26%).  The  unique  properties  of  these  marine  spore- 
formers  are  the  stimulation  of  germination  by  Na+,  the  requirement  of  NH4+  for  germination,  and  the 
ability  of  sporulating  cells  to  concentrate  Ca++  in  a  high  Mg++  environment. 

A  comparative  study  ofanoxic  decomposition  in  salt  and  freshwater  marshes.  JOANNE 
WILLEY  AND  ROBERT  W.  HOWARTH  (Ecosystems  Center,  Marine  Biological 
Laboratory). 

Anoxic  decomposition  represents  75-90%  of  total  sediment  metabolism  in  saltmarsh  systems.  The 
importance  of;;;,  mbic  degradation  in  freshwater  marshes  is  less  well  known.  Anaerobic  mineralization 
of  organic  matter  ,  said  to  proceed  at  a  uniformly  rate  than  aerobic.  However,  anoxic  decomposition 
can  be  more  rapid  than  aerobic  under  some  circumstances.  Most  energy  from  anaerobically  decayed 
organic  substrate  is  conserved  as  inorganic  endproducts  (sulfides  or  methane).  The  resulting  low  assim- 
ilatory  efficiencies  dictate  that  more  organic  substrate  (relative  to  aerobic  decomposition)  is  degraded  to 
maintain  a  given  microbial  biomass.  Thus,  nutrient  limitation  of  decomposition  is  less  likely  anaerobically 
than  aerobically. 

To  evaluate  belowground  decomposition  in  salt  and  freshwater  marshes,  litter  bags  filled  with  one 
of  four  substrates  were  buried  in  May  1982  at  three  depths,  5,  15,  and  25  cm,  in  the  acidic  sediment  of 
a  tidal  freshwater  marsh  at  North  River  and  the  reduced  sediment  at  Great  Sippewissett  Salt  Marsh. 


ECOLOGY  371 

Substrates  used  were  Typha  (dominant  grass  at  North  River)  roots  and  rhizomes,  Typha  shoots  and 
stems,  and  the  same  structures  of  Spartina  patens  (abundant  at  Sippewissett). 

Replicate  litter  bags  collected  at  4  and  12  weeks  demonstrate  the  importance  of  substrate  quality 
and  general  lack  of  environmental  mediation  for  the  first  3  months  of  decomposition.  Weight  losses 
correlated  with  species  and  structure  but  not  with  burial  depth  or  location.  Decay  rates  ranged  from 
-0.71  ±  0.62  for  Spartina  roots  to  -4.53  ±  0.95  for  Typha  roots.  Nutrient  dynamics  reflected  original 
litter  content.  The  only  samples  to  mineralize  nitrogen,  Typha  shoots  and  stems,  were  also  highest  in 
original  N  concentration  (C/N  =  12.28  vs.  about  1 5  for  both  roots  and  25.56  for  Spartina  stems).  Likewise, 
phosphorus-rich  Typha  components  lost  phosphorus,  while  phosphorus-poor  Spartina  immobilized  P. 
Control  of  degradation  as  determined  by  marsh  or  depth  of  incubation  may  become  more  evident  in  the 
remaining  nine  months  as  litter  composition  becomes  less  variable. 

Microbial  colonization  of  filter  paper  incubated  in  saltmarsh  sediments  as  observed 
by  scanning  electron  microscopy.  N.  WOGRIN  (University  of  Massachusetts), 

J.  S.  POINDEXTER,  AND  E.  P.  GREENBERG. 

Anaerobic  cellulose  decomposition  in  sediments  of  the  Great  Sippewissett  Marsh  and  the  School 
Street  Marsh,  Massachusetts,  was  studied  using  scanning  electron  microscopy  (SEM)  of  filter  paper 
incubated  in  situ.  Pieces  of  filter  paper  were  sandwiched  between  microscope  slides  and  implanted  in  the 
top  five  centimeters  of  anaerobic  marsh  sediment.  Filter  paper  samples  were  removed  for  observation 
after  incubation  periods  of  3,  9,  2 1 ,  and  29  days.  Bacterial  colonization  of  the  filter  paper  incubated  in 
the  South  Street  Marsh  sediment  was  apparent  after  nine  days  of  incubation.  Each  microcolony  consisted 
of  cells  of  homogeneous  morphology,  and  it  is  assumed  that  the  organisms  that  persisted  through  prep- 
aration of  the  filter  paper  for  SEM  were  those  that  were  capable  of  stable  attachment  to  the  cellulose 
fibers.  Morphotypes  occurring  in  the  microcolonies  included  ring-shaped  cells  (0.5  ^m  cell  diameter), 
horseshoe  and  helical  cells  (0.5  /^m  cell  diameter),  and  long  rods  (5-8  ^m  X  0.5  nm)  that  appeared  to 
be  flexible.  Cells  of  each  of  these  types  had  rounded  poles,  and  in  many  microcolonies  terminal  swellings 
suggestive  of  endospores  were  observable.  Given  the  anaerobic  conditions  of  incubation,  the  presence 
of  such  terminal  swellings  suggests  that  these  organisms  are  clostridia.  Spirochete-like  organisms  and 
microcolonies  of  cocci  were  also  occasionally  observed.  Cell  types  similar  to  those  detected  by  SEM  were 
observed  when  samples  of  the  incubated  filter  paper  were  stained  with  acridine  orange  and  viewed  with 
epifluorescence  microscopy.  In  addition  to  the  morphologies  described  above,  this  technique  revealed 
the  presence  of  motile  cells:  vibrios,  spirilla,  and  spirochete-like  organisms.  These  were  not  attached  to 
the  filter  paper  and  may  have  been  lost  during  preparation  for  SEM.  By  the  combined  application  of  two 
types  of  microscopy,  two  populations  were  found  associated  with  the  cellulose  during  its  deterioration 
under  anaerobic  conditions:  microcolonies  that  appeared  to  have  a  stable  attachment  to  the  cellulose 
fibers,  and  non-adhering,  motile  associates. 

Parts  of  this  research  were  supported  by  the  Foundation  for  Microbiology,  NASA  NAGW-306,  and 
the  Dept.  of  Energy  DEFG  02  82ER 12079. 

FERTILIZATION  AND  DEVELOPMENT 

Stimulus/response  coupling  in  sponge  aggregation:  evidence  for  calcium  as  an  in- 
tracellular  messenger.  CATHLEEN  ANDERSON,  ABBY  M.  RICH,  ADAM  DICKER, 
PHILIP  DUNHAM,  AND  GERALD  WEISSMANN  (Marine  Biological  Laboratory). 

Aggregation  of  dissociated  sponge  cells  (Microciona  prolifera)  has  been  proposed  as  a  model  for  cell- 
cell  recognition  mediated  by  a  specific  proteoglycan  aggregation  factor  (MAF).  To  test  whether  sponge 
cells  undergo  stimulus/response  coupling  in  which  intracellular  Ca++  is  a  messenger,  mechanically  dis- 
sociated cells  were  studied  in  a  Payton  aggregometer  conventionally  employed  for  kinetic  analysis  of 
aggregation  of  platelets  and  neutrophils.  Changes  in  light  transmission  paralleled  aggregation  as  judged 
by  light,  scanning,  and  transmission  electron  microscopy  (EM).  Cells  (2  X  108/ml)  were  equilibrated  (30- 
60  min)  in  Ca++-,  Mg++-free  sea  water  (pH  7.8)  with  EDTA  to  deplete  cells  of  Ca++  and  to  inactivate 
soluble  MAF.  In  the  presence,  but  not  absence,  of  Ca++  (>5  mM)  partially  purified  MAF  (from  Dr.  M. 
Burger)  aggregated  both  living  and  glutaraldehyde-fixed  cells.  MAF  remained  associated  with  the  surface 
of  EDTA-treated  cells  judged  by  their  aggregation  in  response  to  anti-MAF,  but  not  pre-immune  serum. 
Evidence  for  a  messenger  role  of  intracellular  Ca++  was  the  following:  1 )  Addition  of  Ca++  (>2.5  mM) 
to  Ca++-depleted  cells  induced  aggregation  that  varied  directly  with  the  Ca++  concentration.  2)  Addition 
of  calcium  ionophores  (A23187,  ionomycin;  >5  ^M)  caused  aggregation  which  varied  with  extracellular 
Ca++  and  far  exceeded  that  provoked  by  Ca++  alone.  Glutaraldehyde-fixed  cells  did  not  respond  to 
ionophores  ±  Ca.  3)  Calcium  antagonists  inhibited  aggregation.  These  included  a  napthalene  sulphon- 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 


amide  inhibitor  of  the  Ca-calmodulin  complex  (W-7;  >15  nM),  a  calcium  channel  blocker  (verapamil; 
>100  nM)  and  three  non-steroidal  anti-inflammatory  agents  (indomethacin,  ibuprofen,  piroxicam;  >50 
nM).  Cells  remained  viable  in  all  circumstances  (Trypan  Blue  exclusion;  supravital  staining;  transmission 
EM).  Results  indicate  that  early  events  (0-5  min)  of  sponge  aggregation  can  be  quantified  by  a  continuous 
recording  technique,  and  that  it  is  not  simply  the  passive  response  of  an  inert  cell  to  an  extracellular 
proteoglycan.  Rather,  the  sponge,  like  the  platelet  or  neutrophil,  recognizes  surface  ligands  to  which  it 
responds  by  calcium-dependent  stimulus-response  coupling. 

The  effects  ofquercetin  and  ionophore  A23187  on  meiosis  initiation  in  Spisula  and 
Asterias  oocytes.  WILLIAM  R.  ECKBERG  (Department  of  Zoology,  Howard  Uni- 
versity, Washington,  D.  C.  20059). 

To  examine  the  roles  of  calcium  and  calcium  sequestration  in  meiosis  initiation,  we  treated  oocytes 
of  Spisula  and  Asterias  with  quercetin,  an  ATPase  inhibitor.  Isotonic  Cad?  initiated  GVBD  in  Spisula 
oocytes  only  in  the  presence  of  quercetin.  This  suggests  that  quercetin  initiates  GVBD  by  inhibition  of 
calcium  sequestration.  However,  when  the  oocytes  were  treated  in  sea  water,  meiosis  was  not  initiated, 
even  when  excess  KC1  was  added  in  amounts  below  the  threshold  for  parthenogenesis.  High  doses  of 
quercetin  blocked  GVBD  but  not  fertilization  envelope  elevation  when  they  were  induced  by  ionophore 
or  excess  KC1.  Therefore  the  drug  can  have  an  additional  inhibitory  effect  on  GVBD.  Kinetic  studies 
showed  that  quercetin  inhibited  a  relatively  early  event  in  GVBD  induction.  Quercetin  also  inhibited 
fertilization. 

When  starfish  oocytes  were  treated  with  relatively  high  concentrations  ofquercetin,  1  -methyl  adenine 
(1-MA)  stimulation  of  GVBD  was  blocked.  When  1-MA  was  absent  or  present  in  subthreshold  concen- 
trations, lower  concentrations  ofquercetin  stimulated  meiosis  somewhat. 

Treatment  of  Spisula  oocytes  with  ionophore  resulted  in  egg  activation  as  demonstrated  by  fertil- 
ization envelope  elevation,  GVBD,  and  polar  body  formation.  This  result  was  dependent  upon  extra- 
cellular calcium.  This  result,  together  with  the  quercetin  results,  suggests  that  intracellular  calcium  in 
Spisula  oocytes  is  exchangeable.  Ionophore  activated  Asterias  oocytes  as  shown  by  fertilization  envelope 
elevation,  but  failed  to  initiate  GVBD. 

These  results  further  indicate  the  importance  of  calcium  sequestration  in  the  maintenance  of  the 
germinal  vesicle,  but  they  also  show  that  quercetin  can  have  other  effects  on  GVBD  than  the  stimulatory 
effect  previously  shown. 

On  the  role  of  maternal  mRNA  in  sea  urchins:  studies  of  a  protein  which  appears 
to  be  destroyed  at  a  particular  point  during  each  cell  division  cycle.  TOM  EVANS, 
TIM  HUNT,  AND  JIM  YOUNGBLOM  (Physiology  Course,  MBL). 

We  have  reinvestigated  the  pattern  of  protein  synthesis  after  activation  of  eggs  of  the  sea  urchin 
Arbacia  punctulata  with  sperm,  NH4C1,  or  A23187.  Eggs  were  labeled  continuously  with  35S-methionine, 
samples  taken  every  10  minutes,  and  the  pattern  of  protein  synthesis  analyzed  on  SDS-polyacrylamide 
gels.  Autoradiography  of  these  gels  revealed  a  heavily  labeled  protein,  Mr  55,000,  which  showed  striking 
behavior  as  development  proceeded:  each  time  the  eggs  divided,  it  disappeared.  Its  synthesis  is  barely 
detectable  in  unfertilized  eggs,  although  they  contain  high  levels  of  mRNA  for  this  protein,  which  we  call 
"cyclin."  Cyclin  has  the  following  additional  properties:  (  1  )  It  disappears  completely  after  inhibition  of 
protein  synthesis  by  emetine.  (2)  It  is  synthesized  at  a  constant  rate  as  measured  by  successive  10-minute 
pulses  with  35S-methionine  during  the  first  100  minutes  of  development.  (3)  Cyclin  does  not  bind  to  a 
monoclonal  anti-tubulin  antibody  which  reacts  strongly  with  Arbacia  tubulin.  (4)  In  the  presence  of 
inhibitors  of  cell  division,  10~4  M  colchicine,  10  5  M  taxol,  or  4  X  10~6  M  cytochalasin  D  the  level  of 
cyclin  rose  normally,  but  disappeared  very  slowly.  (5)  Activation  of  protein  synthesis  with  10  mA/  NH4C1 
led  to  the  continuous  accumulation  of  cyclin  with  no  sign  of  breakdown  over  a  period  of  2  hours.  In 
contrast,  A23187  gave  a  pattern  of  cyclin  synthesis  and  breakdown  very  similar  to  that  produced  in  the 
presence  of  the  inhibitors  mentioned  above  in  (3). 

Preliminary  experiments  show  that  proteins  which  exhibit  similar  properties  are  found  in  the  urchin 
Lytechinus  pictus  and  the  clam  Spisula  solidissima. 

This  work  was  supported  by  NIH  training  grant  GM-3  1  1  36-04  to  the  Physiology  Course.  We  thank 
John  Kilmartin  for  a  gift  of  antibody,  and  Amersham  International  for  donating  the  labeled  methionine. 

The  ontogeny  of  the  fertilization  site  in  Hydractinia  echinata  (hydrozoa).  GARY 
FREEMAN  (University  of  Texas  at  Austin). 

Sperm  will  only  fuse  with  hydrozoan  eggs  at  the  site  of  polar  body  formation  (Freeman  and  Miller 
1982,  Develop.  Biol.  in  press).  This  suggests  that  there  is  a  special  cell  surface  and/or  cell  membrane 
differentiation  at  this  site. 


FERTILIZATION  AND  DEVELOPMENT  373 

The  time  during  oocyte  maturation  when  this  site  forms  was  established  by  adding  sperm  to  oocytes 
which  are  at  different  stages  of  the  process  of  maturation.  After  a  5  minute  exposure  to  sperm,  those 
sperm  that  had  not  been  incorporated  into  the  oocyte  were  destroyed  with  a  0.0005%  SDS  solution.  The 
process  of  maturation  was  allowed  to  go  to  completion  and  sperm-egg  fusion  was  assayed  by  establishing 
whether  or  not  these  eggs  cleaved.  Only  eggs  treated  with  sperm  after  second  polar  body  formation 
cleaved. 

The  role  of  the  oocyte  nucleus  in  setting  up  the  fertilization  site  was  examined  by  moving  the 
germinal  vesicle  or  meiotic  apparatus  to  a  new  position  at  different  stages  of  oocyte  maturation  and 
establishing  whether  the  displacement  of  the  nucleus  also  displaced  the  site  of  fertilization.  This  was  done 
by  centrifuging  the  oocytes  at  3000  X  g.  Prior  to  centrifugation  the  initial  position  of  the  oocyte  nucleus 
was  marked  with  the  vital  dye  nile  blue  A.  Centrifugation  stratifies  the  contents  of  the  oocyte  into  a 
centripetal  pigment  zone,  a  clear  zone,  and  a  centrifugal  yolk-filled  zone.  The  oocyte  nucleus  is  always 
found  in  the  clear  zone  just  below  the  pigment  layer.  Those  oocytes  were  selected  where  the  mark  and 
the  pigment  cap  were  not  congruent.  After  the  process  of  maturation  was  completed  the  oocyte  was  cut 
into  fragments  in  such  a  way  that  one  fragment  contained  the  stain  mark  and  the  other  fragment  contained 
the  pigment  zone  and  most  of  the  clear  cytoplasm.  Sperm  was  added  to  each  fragment  and  fertilization 
was  assayed  by  monitoring  cleavage.  One  can  move  the  site  of  fertilization  by  changing  the  position  of 
the  nucleus  at  any  time  prior  to  second  polar  body  formation.  After  this  time  period  the  site  of  fertilization 
is  fixed. 

Supported  by  grant  GM  20024  from  the  National  Institutes  of  Health. 

An  unexpectedly  steep  developmental  gradient  in  Asterias  forbesi  embryos  induced 
by  anoxia.  S.  INOUE,  S.  B.  POTREBIC,  C.  R.  BROWN,  AND  D.  A.  LUTZ  (Marine 
Biological  Laboratory). 

Fertilized  eggs  from  the  starfish  Asterias  forbesi,  sandwiched  between  slide  and  coverslip,  crowded 
into  a  monolayer  in  filtered  sea  water  and  surrounded  by  a  sealed  air  space,  generate  a  steep  developmental 
gradient.  Observed  ca.  1 5  hours  after  fertilization,  a  culture  drop  with  diameter  greater  than  5  mm  and 
density  greater  than  19  embryos  per  mm2  has  fertilized  eggs  in  the  center  which  have  not  cleaved.  From 
the  center  outward  are  concentric  rings  of  cells  which  have  divided  1,  2,  3  .  .  .9  times.  Even  in  a  single 
approx.  1 50  ^m  diameter  embryo,  cells  on  the  inside  may  be  arrested  two  division  cycles  earlier  than 
the  outer  cells.  The  wave  of  division  arrest  is  propagated  outward  radially  at  a  rate  of  approximately  60 
nm  per  hour.  Finally  one  reaches  an  outside  rim  of  1.5-2.2  mm  in  which  all  embryos  have  hatched  as 
swimming  blastulas  simultaneously  with  controls. 

Anoxia  was  determined  to  cause  the  steep  developmental  gradient  by  the  following  experiments.  1) 
In  a  crowded  hanging  drop  preparation  no  gradient  developed.  2)  Removing  CO2  with  KOH  did  not 
reduce  the  gradient.  3)  If  O2  is  continuously  perfused  across  the  microdrop  preparation,  no  gradient 
develops.  4)  When  a  preparation  is  made  containing  0.1%  methylene  blue,  the  dye  is  quickly  reduced 
to  its  colorless  form  except  in  a  1  mm  ring  along  the  outer  edge. 

Even  after  long  periods  of  anoxic  arrest,  the  embryos  in  the  center  of  the  gradient  do  not  lyse  and 
can  be  revived  by  introduction  of  O2.  They  then  undergo  successive  divisions  and  develop.  The  appearance 
of  such  a  sharp  gradient  of  anoxic  arrest  and  its  reversibility  suggest  that  self-generated  redox  gradients 
may  well  affect  differentiation  and  development  in  embryos  and  tissues. 

Supported  by  U.  of  PA  General  Honors  Program,  grants  NSF  PCM  79-22136  and  NIH  7R01- 
GM31617  (S.I.),  and  NSERC  post  grad.  fellowship  (D.A.L.). 

Colcemid  but  not  cytochalasin  inhibits  asymmetric  nuclear  positioning  prior  to  un- 
equal cell  division.  DOUGLAS  A.  LUTZ  AND  SHINYA  INOUE  (Marine  Biological 
Laboratory). 

We  investigated  the  mechanism  of  nuclear  migration  and  spindle  orientation  that  precedes  unequal 
cleavage  by  interfering  with  microtubule  and  actin-microfilament  assembly.  In  echinoid  embryos,  the 
nucleus  and  spindle  are  positioned  asymmetrically  and  oriented  appropriately  prior  to  the  unequal  macro- 
mere/micromere  forming  division.  Upon  completion  of  the  3rd  division,  the  vegetal  blastomere  nuclei 
of  Lvtcchinus  variegatus  migrate  from  a  central  position  to  the  vegetal  pole  (VP)  cortex  at  a  rate  of  1.5- 
2.0  /nm/min;  a  similar  migration  was  also  observed  in  vegetal  blastomeres  of  Clypeaster  and  Hemicentrotus 
by  Dan  (1979,  Dev.  Growth  and  Diff.  21:  527-535).  In  Lytechinus,  the  nucleus  travels  approximately  20 
jim  in  12  min  at  21  °C  to  a  position  5-8  ^m  from  the  VP  cortex.  There  the  nuclear  envelope  breaks 
down  and  the  mitotic  spindle  forms,  already  properly  oriented.  In  addition  to  positional  asymmetry, 
morphological  asymmetry  is  present  within  the  mitotic  spindle;  the  peripheral  aster  is  truncate  whereas 
the  internal  aster  is  radiate.  When  Colcemid,  which  disassembles  mitotic  microtubules  in  vivo,  is  applied 
at  the  completion  of  the  3rd  cleavage  in  concentrations  greater  than  1  nM,  the  vegetal  blastomere  nuclei 


374  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

do  not  migrate  but  remain  centrally  situated.  If,  at  nuclear  envelope  breakdown,  Colcemid  is  washed  out 
and  inactivated  within  the  embryo  by  a  short  exposure  to  366  nm  illumination  (Aronson  and  Inoue 
1970,  J.  Cell  Biol.  45:  470-477),  the  spindle  forms  centrally  and  with  symmetric  asters.  Anaphase  ensues 
before  appreciable  spindle  migration,  so  the  division  tends  to  produce  daughter  blastomers  of  nearly 
equal  size.  Concentrations  of  cytochalasins  D(20  nAf),  E(  1  nM)  and  dihydroxyB  ( 1  nAf),  which  interfere 
with  actin  assembly  and  relax  cleavage  furrows  within  3  min,  had  no  effect  on  nuclear  migration;  the 
rate  of  nuclear  migration,  final  distance  from  VP  cortex,  and  astral  asymmetry  are  similar  to  controls. 
These  data  suggest  that  a  microtuble-based  or  -mediated,  but  not  an  actin  microfilament-based 
or  -mediated  motile  system  is  responsible  for  the  nuclear  migration  to  its  asymmetric  position. 

Supported  by  NSERCC  post-graduate  fellowship  to  D.A.L.  and  NIH  7R01-GM31617  and  NSF 
PCM  79-22136  to  S.I. 

The  role  of  the  germinal  vesicle  in  the  1-methyladenine-induced  changes  in  protein 
synthesis  in  Asterias  oocytes.  MARK  Q.  MARTINDALE  AND  BRUCE  P.  BRAN- 
DHORST  (McGill  University). 

Asterias  oocytes  undergo  dramatic  translationally  mediated  qualitative  and  quantitative  changes  in 
protein  synthesis  after  induction  of  meiotic  maturation  by  1-methyladenine  (IMA)  (Rosenthal  el  al. 
1982,  Develop.  Biol.  91:  215-220).  The  beginning  of  these  changes  coincides  with  the  breakdown  of  the 
germinal  vesicle  (GV).  To  investigate  the  role  of  the  release  of  GV  contents  in  these  changes  in  protein 
synthesis  we  isolated  nucleated  and  enucleated  fragments  of  Asterias  oocytes.  Oocytes  were  collected  in 
Ca++-free  sea  water  (CFSW  pH  5.0)  and  filtered  through  cheesecloth  to  remove  follicle  cells  and  jelly 
coats.  The  oocytes  were  then  layered  on  a  discontinuous  sucrose  gradient  consisting  of  an  upper  1.5:1 
( 1  M  sucrose:  CFSW)  layer,  a  middle  4: 1  layer,  and  a  1  M  sucrose  cushion.  The  samples  were  centrifuged 
at  5000  rpm  for  20  min  at  8-10°C  in  a  Beckman  JA-13  swinging  bucket  rotor.  The  speed  was  then 
increased  to  12,000  rpm  for  the  last  20  min.  Enucleated  fragments  were  collected  off  of  the  1  M  cushion 
and  their  purity  established  by  phase  contrast  microscopy.  Nucleated  fragments  were  taken  from  the  top 
of  the  4: 1  layer.  All  fragments  were  washed  with  filtered  sea  water  and  aliquots  activated  by  addition  of 
3.0  X  10~5  M  IMA.  Fragments  and  intact  oocytes  were  labeled  with  35S-methionine  (0.5  mCi/ml;  1200 
Ci/mMol)  for  30  min  and  newly  synthesized  proteins  compared  by  electrophoresis  on  10%  polyacrylamide 
gels  containing  SDS.  Autoradiographs  show  that  the  proteins  synthesized  by  both  unactivated  nucleated 
and  enucleated  fragments  were  indistinguishable  from  those  of  intact  oocytes.  Following  addition  of  1  MA 
essentially  identical  changes  in  protein  synthesis  were  observed  for  nucleated  and  enucleated  fragments 
as  well  as  activated  intact  oocytes.  We  conclude  that  maternal  RNAs  or  translational  factors  required 
for  the  changes  in  protein  synthesis  are  not  sequestered  in  the  GV.  Thus,  most,  or  all,  of  the  maternal 
mRNAs  becoming  available  for  translation  during  maturation  are  stored  in  the  cytoplasm  of  the  oocyte. 

This  research  was  carried  out  in  the  Embryology  Course  which  is  supported  by  a  grant  from 
the  N.I.H. 

Effect  ofgossypol  on  Arbacia  sperm  A  TPase.  HIDEO  MOHRI  (Department  of  Biology, 
University  of  Tokyo,  Japan),  KYOKO  MATSUDA,  S.  S.  KOIDE,  AND  SHEL- 
DON J.  SEGAL. 

Motility  of  Arbacia  spermatozoa  is  inhibited  by  gossypol,  and  both  pyruvate  dehydrogenase  and 
Mg2+-ATPase  activities  of  the  sperm  mitochondria  are  much  reduced  by  this  substance  (Adeyemo  et  al. 
1981,  Biol.  Bull.  161:  333),  suggesting  that  gossypol  limits  ATP  supply  to  the  motility  apparatus  of  the 
spermatozoa.  The  present  study  determined  whether  or  not  gossypol  directly  affects  the  motility  system 
of  sea  urchin  spermatozoa. 

Intact  Arbacia  spermatozoa  immediately  stop  their  movement  when  exposed  to  0.3  mM  gossypol. 
At  lower  concentrations  the  effect  is  less  pronounced.  The  ATP-induced  motility  of  sperm  demembranated 
with  Triton  X-100  is  relatively  insensitive  to  the  action  ofgossypol.  To  achieve  complete  arrest  of  motility 
concentrations  ofgossypol  as  high  as  1  mM  are  required.  The  ATP-induced  motility  of  demembranated 
sperm  exposed  to  0.3  mM  gossypol  is  as  vigorous  as  that  displayed  by  controls.  When  the  spermatozoa 
are  preincubated  in  0.3  mM  gossypol  for  10  min,  and  subjected  subsequently  to  the  demembranation 
and  reactivation  procedure,  the  demembranated  sperm  become  vigorously  motile.  This  result  supports 
the  postulate  that  gossypol  limits  the  ATP  supply  to  the  sperm's  motility  apparatus. 

To  determine  whether  gossypol  directly  influences  dynein  ATPase  activity,  Arbacia  spermatozoa 
were  fractionated  into  the  head-plus-midpiece  and  tail  fractions.  The  tails  were  further  demembranated 
to  obtain  the  axonemes.  Mg2+-ATPase  activities  of  all  these  fractions  are  inhibited  by  gossypol.  At  a 
concentration  of  about  50  nM,  gossypol  inhibits  the  enzymatic  activities  of  these  preparations  by  50%. 
Finally,  2  IS  dynein  was  extracted  from  Arbacia  sperm  axonemes  and  the  effect  ofgossypol  on  its  Mg2+- 
ATPase  activity  was  tested.  The  purified  2 1 S  dynein  ATPase  is  inhibited  by  gossypol  at  a  concentration 


FERTILIZATION  AND  DEVELOPMENT  375 

of  2  \iM.  Thus,  although  gossypol  would  primarily  affect  the  ATP-generating  system  in  vivo,  it  also 
inhibits  dynein  ATPase. 

Supported  by  the  Rockefeller  Foundation  and  NIH.  H.M.  is  the  Rand  Lecturer  at  MBL  in  1982. 

Effect  of  heat  shock  on  nuclear  RNP  structure  in  mammalian  cells.  CHRISTINE 
MAUTE  MORGANELLI  (Dartmouth  College). 

Heterogeneous  nuclear  RNA  is  normally  complexed  with  a  specific  set  of  proteins,  forming  ribo- 
nucleoprotein  particles  termed  hnRNP.  These  particles  are  likely  to  be  involved  in  mRNA  processing. 
Recently,  it  has  been  shown  in  cultured  Drosophila  cells  that  the  assembly  of  hnRNA  into  hnRNP 
particles  is  blocked  by  heat  shock  (S.  Mayrand  and  T.  Pederson,  personal  communication).  Because 
mammalian  cells  also  show  a  heat  shock  effect  on  protein  synthesis,  it  was  of  interest  to  determine 
whether  hnRNP  assembly  is  also  altered  in  these  cells  by  elevated  temperature.  HeLa  or  mouse  eryth- 
roleukemia  cells  were  pulse-labeled  with  3H-uridine  at  37 °C  or  the  desired  elevated  temperature,  and 
hnRNP  particles  were  isolated  from  nuclei  by  standard  procedures.  The  protein  content  of  these  particles 
was  analyzed  by  equilbrium  centrifugation  in  Cs"2SO4  density  gradients.  Heat  shock  altered  the  assembly 
of  hnRNP  in  both  HeLA  and  mouse  erythroleukemia  cells.  HnRNP  from  control  cells  (37°C)  banded 
at  a  density  of  1.35  g/cm3  (approx.  80%  protein:  20%  RNA),  whereas  after  heat  shock  (39°-43°C)  an 
increasing  proportion  of  the  particles  banded  at  higher  densities  (1.45-1.60  g/cm3),  indicative  of  a  greatly 
reduced  protein  content.  Further  results  indicate  that  the  shift  in  hnRNP  structure  is  gradual  at  pro- 
gressively higher  temperatures,  rather  than  an  all-or-none  response.  The  effect  of  heat  shock  on  hnRNP 
is  first  observed  at  39°C,  whereas  no  inhibition  of  total  hnRNA  transcription  occurs  until  42°-43°C. 
The  possibility  arises  that  the  blocked  hnRNP  assembly  is  related  to  altered  post-transcriptional  mRNA 
processing  after  heat  shock.  In  particular,  this  condition  might  favor  the  processing  of  mRNA's  that  do 
not  undergo  splicing.  It  is  noteworthy  that  most  of  the  heat  shock  mRNA's  lack  intervening  sequences. 

This  work  was  supported  by  N.I.H.  training  grant  GM-31 136-04  to  the  Physiology  Course.  I  thank 
Sandra  Mayrand  and  Thoru  Pederson  for  their  expert  advice  and  guidance,  and  Tim  Hunt  for  his  untiring 
help  in  the  lab. 

Synthesis  of  5S  RNA  and  tRNA  in  cleaving  sea  urchin  embryos:  effect  of  altering 
cell  interactions.  ANNE  F.  O'MELIA  (Department  of  Biology,  George  Mason 
University,  Fairfax,  VA  22030). 

The  synthesis  of  5S  RNA  and  of  transfer  RNA  (tRNA)  has  been  shown  to  occur  as  early  as  the  16- 
to  32-cell  stage  in  cleaving  sea  urchin  embryos  (O'Melia  1979,  Develop.  Growth  and  Differ.  21:  99-108). 
Rates  of  accumulation  of  newly  made  5S  RNA  and  tRNA  per  cell  are  highest  during  cleavage  and  decline 
about  threefold  during  development  to  the  pluteus  stage  (O'Melia  1979,  Differentiation  15:  97-105).  The 
present  study  determined  whether  normal  cell  associations  and  interactions  are  necessary  for  5S  RNA 
and  tRNA  synthesis  in  cleaving  embryos  of  the  sea  urchin,  Arbacia  punctulata.  Cell  interactions  were 
altered:  ( 1 )  by  culturing  cleaving  embryos  in  evans  blue,  which  induces  animalization  (ectodermalization), 
and  in  LiCl,  which  induces  vegetalization  (endo-mesodermalization)  of  whole  sea  urchin  embryos;  and 
(2)  by  culturing  cells  dissociated  from  cleaving  embryos  under  conditions  which  prevent  reaggregation. 
Control  and  experimental  embryos  and  dissociated  cells  each  were  labeled  from  3  h  to  6  h  post  fertilization 
with  guanosine-[8-3H]  and  with  L-[3H-methyl]-methionine.  Total  cellular  RNA  was  extracted  using  the 
cold  (4°C)-phenol-sodium  dodecyl  sulfate  method,  and  purified  (LiCl-soluble)  RNA  preparations  were 
fractionated  by  electrophoresis  on  10%  polyacrylamide  gels.  Rates  of  accumulation  of  newly  made  5S 
RNA  and  of  tRNA  in  control  and  in  experimental  embryos  were  calculated  from  the  radioactivity 
coincident  with  the  5S  RNA  and  with  the  tRNA  absorbance  peaks  (A260  nm)  on  each  gel,  from  the 
known  GMP  composition  of  sea  urchin  5S  RNA  and  tRNA,  and  from  the  average  specific  radioactivity 
of  the  GTP  precursor  pool  during  the  3-h  labeling  period.  The  results  show  that  rates  of  synthesis  of  5S 
RNA  and  tRNA  per  embryo  and  per  cell  are  similar  in  control  embryos  and  in  cleaving  embryos  cultured 
in  the  presence  of  animalizing  and  vegetalizing  agents.  In  addition,  cells  dissociated  from  cleavage  embryos 
retained  the  ability  to  synthesize  5S  RNA  and  tRNA.  These  results  suggest  that  normal  cell  associations 
and  interactions  are  not  necessary  for  the  synthesis  of  5S  RNA  and  tRNA  in  cleaving  sea  urchin  embryos. 

[Support:  CRAS,  GMU.] 

Vitellogenesis  in  the  hepatopancreas  and  ovaries  ofCarcmus  maenas.  JEANNE  E. 
PAULUS  AND  HANS  LAUFER  (The  Biological  Sciences  Group,  The  University  of 
Connecticut,  Storrs,  CT). 

The  site(s)  of  synthesis  of  yolk  proteins  or  their  precursors  has  never  been  clarified  in  Crustacea.  It 
has  been  shown  repeatedly  that  removing  eyestalks  of  various  crustaceans  during  their  reproductive 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

season  stimulates  ovarian  growth  and  presumably  vitellogenesis.  Eyestalk  ablation  of  mature  female  C. 
maenas  during  May,  June,  and  early  July  increased  the  mean  gonadal  index  from  1.1  to  4.0%,  and  the 
percentage  of  spawning  females  rose  to  50%  relative  to  30%  in  unoperated  controls.  In  controls  75%  were 
non-vitellogenic,  while  90%>  of  the  experimental  group  were  vitellogenic  36  days  after  eyestalk  removal. 

Since  potential  sites  of  action  of  the  ovary  stimulating  factor  are  the  ovary  and  hepatopancreas,  we 
have  developed  an  in  vitro  system  for  assaying  lipovitellin  synthesis  in  these  possible  target  tissues. 
Fragments  of  tissue  are  cultured  in  media  consisting  of  1.5  mA/  D-glucose,  salts,  antibiotics,  and  3H-L- 
amino  acids  at  17°C  up  to  16  hours.  The  percentage  lipovitellin  synthesized,  relative  to  total  TCA- 
precipitable  counts,  is  assayed  by  a  double  immunoprecipitation  technique  using  lipovitellin-specific 
antibody  produced  in  rabbits,  and  protein  A  of  Staph ylococc us  aureus  (Cowen  I  strain). 

There  is  a  developmental  pattern  of  lipovitellin  synthesis  in  the  hepatopancreas  and  ovary.  The 
hepatopancreas  is  most  active  in  lipovitellin  production  during  stage  3,  when  oocytes  measure  0.2-0.4 
mm  in  diameter.  The  synthesis  of  lipovitellin  is  greatest  in  the  ovary  at  stage  4,  characterized  by  oocytes 
0.4-0.7  mm  in  diameter.  The  activity  of  the  hepatopancreas  is  relatively  low  at  this  time. 

This  is  the  first  report  demonstrating  conclusively  the  synthesis  of  lipovitellin,  or  its  precursors,  in 
the  hepatopancreas.  Furthermore,  this  synthesis  coincides  with  the  time  when  the  ovary  incorporates 
serum  vitellogenins  into  developing  oocytes.  Before  and  after  this  stage  of  specific  uptake,  the  ovary  is 
the  major  contributor  of  lipovitellins  to  the  oocyte. 

This  research  was  supported  in  part  by  grants  from  the  National  Science  Foundation,  the  Institute 
of  Water  Resources,  and  The  University  of  Connecticut  Research  Foundation. 

A  new  met  hod  for  preparing  marine  eggs  for  microinjection:  the  "fly  paper ' '  technique. 
MARK  BENNETT  POCHAPIN,  JEAN  M.  SANGER,  AND  JOSEPH  W.  SANGER  (De- 
partment of  Anatomy  G/3,  University  of  Pennsylvania  School  of  Medicine, 
Philadelphia,  PA  19104). 

A  simple  method  for  microinjecting  large  numbers  of  sea  urchin  eggs  was  devised  by  attaching  eggs 
to  a  poly-L-lysine-coated  coverslip  and  placing  it  in  an  open  Petri  dish  containing  sea  water.  The  eggs 
can  be  fertilized  either  before  or  after  they  are  attached  to  the  coverslip  and  will  divide  normally,  hatch, 
and  form  plutei.  In  this  way,  specially  constructed  chambers  are  not  required  and  the  eggs  can  be 
microinjected  from  the  top  in  the  same  manner  as  tissue  culture  cells.  The  seawater  medium  can  be 
removed  easily  and  replaced  with  artificial  medium  if  desired,  or  the  coverslip  can  be  removed  from  the 
dish  and  mounted  in  order  to  flatten  the  cells  for  better  visualization  of  the  spindle.  Preparation  of 
coverslips  requires  that  they  be  thoroughly  cleaned  in  detergent  and  distilled  water,  then  in  95%  ethanol 
followed  by  vigorous  rubbing  with  cheesecloth  until  the  coverslip  surface  feels  smooth.  Approximately 
20  drops  of  a  freshly  prepared  poly-L-lysine  (Sigma  Chemical  Co.,  mol  wt  >  300,000)  solution  (1  mg/ 
ml)  are  added  to  the  cleaned  coverslips  and  allowed  to  stand  for  one  hour.  The  coverslips  are  drained 
by  touching  one  edge  to  a  piece  of  filter  paper  and  then  air-dried  and  placed  in  35-mm  plastic  Petri 
dishes.  Eggs  of  Arbacia  punctulata,  shed  into  filtered  sea  water,  were  transfered  with  a  Pasteur  pipette 
to  form  a  large  drop  on  the  coated  coverslip.  Within  five  minutes,  the  eggs  settled  from  the  drop  and 
adhered  to  the  coverslip,  after  which  time  more  sea  water  was  added  to  half-fill  the  Petri  dish.  The 
attached  eggs  could  be  fertilized  in  the  dish  by  adding  a  drop  of  sperm  suspended  in  sea  water.  Fertilization 
membranes  formed  normally  and  the  excess  sperm  quickly  stuck  to  the  poly-L-lysine  surface  giving  it 
the  appearance  of  fly  paper.  Microinjection  was  accomplished  by  placing  the  Petri  dish  on  the  stage  of 
an  inverted  microscope  and  injecting  desired  solutions  into  embryos  or  unfertilized  eggs  with  the  aid  of 
a  pressure  regulator  connected  to  a  nitrogen  tank.  When  Lucifer  yellow  was  injected  into  unfertilized 
eggs  that  were  subsequently  fertilized,  development  proceeded  normally  to  the  pluteus  stage  where  all 
cells  contained  Lucifer  yellow.  We  believe  that  this  method  of  microinjecting  sea  urchin  eggs  offers  a 
relatively  simple  way  of  introducing  a  variety  of  agents  into  a  large  number  of  eggs  or  embryos  in  a  short 
period  of  time,  enabling  the  effects  of  the  agents  to  be  monitored  during  development. 

This  work  was  supported  by  funds  from  the  National  Institutes  of  Health.  We  are  grateful  to  the 
Basic  Research  Support  Grant  Committees  of  the  University  of  Pennsylvania  for  funds  for  some  of  the 
video  cameras  and  recorders  used  in  this  work. 

Sperm  agglutinating  factor  isolated  from  Spisula  oocytes.  EIMEI  SATO,  S.  J.  SEGAL, 
AND  S.  S.  KOIDE  (Population  Council). 

A  membrane  component  present  on  the  surface  of  Spisula  oocytes  was  found  to  induce  sperm 
agglutination.  Purification  and  characterization  of  the  oocyte  surface  component  (OSC)  were  carried  out. 
Several  extraction  media  were  tested  at  varying  incubation  times.  The  following  media  were  used:  (A) 
1  A/ urea,  5  mA/EDTA,  10  mA/Tris- HC1,  pH  7.4;  (B)  1  M  urea,  10  mA/Tris- HC1,  pH  7.4;  (C)  5  mA/ 


FERTILIZATION  AND  DEVELOPMENT  377 

EDTA  in  artificial  sea  water  (ASW).  Oocytes  incubated  up  to  15  min  in  media  A  or  B  at  22°C  exclude 
the  dye  trypan  blue  indicating  that  they  are  viable.  After  15  min,  however,  progressive  staining  of  the 
oocytes  occurs.  Oocytes  incubated  in  medium  C  for  two  hours  or  longer  remain  viable.  Thus,  exposure 
of  oocytes  to  media  containing  1  M  urea  for  longer  than  1 5  min  results  in  disruption  of  the  cell  membrane. 

Oocytes  incubated  in  medium  A  or  B,  then  washed  with  400  mM  Tris-  HC1,  pH  7.4,  2  mM  CaCl2 
do  not  undergo  germinal  vesicle  breakdown  (GVBD)  after  exposure  to  sperm.  However,  GVBD  is  induced 
in  these  oocytes  by  exposure  to  70  mM  KC1.  This  suggests  that  urea  treatment  results  in  the  removal 
or  alteration  of  a  membrane  component  involved  in  sperm-oocyte  interaction. 

Sperm  added  directly  to  medium  A,  B  or  C  do  not  agglutinate.  After  oocytes  are  incubated  in 
medium  A  or  B,  the  ambient  medium  induces  sperm  agglutination;  the  clumps  remain  intact  for  at  least 
one  hour  while  the  aggregate  sperm  retain  motility.  This  observation  indicates  that  a  factor  is  extracted 
from  oocytes  which  induces  sperm  agglutination.  The  Spisula  oocyte  extract  does  not  agglutinate  sperm 
ofArbacia,  Asterias,  Ovalipes,  or  Chaetoptems.  The  agglutinating  factor  is  stable  at  100°C  for  15  min 
and  is  not  denatured  by  freeze-drying.  It  forms  a  precipitate  when  dialyzed  against  distilled  water  and 
is  destroyed  by  trypsin.  It  is  precipitated  by  acetone  and  is  not  absorbed  on  charcoal.  These  characteristics 
suggest  that  the  factor  is  a  protein  or  a  glycoprotein.  Its  MW  is  estimated  to  be  about  15  to  25  K  daltons 
on  the  basis  of  gel  filtration  on  Sephadex  G-100  and  by  dialysis  procedures  using  cellulose  tubings  with 
defined  MW  cutoffs. 

When  the  factor  is  purified  by  ammonium  sulfate  fractionation  (30%  saturation)  followed  by  gel 
filtration  on  Sephadex  G-100,  four  major  peaks  are  obtained.  The  fractions  comprising  the  second  and 
third  peaks  possess  sperm  agglutinating  activity  at  a  concentration  of  2.5  Mg/ml. 

Supported  by  the  Rockefeller  Foundation  and  NIH.  E.S.  is  a  postdoctoral  fellow  of  the  Rockefeller 
Foundation. 

Indomethacin,  an  anti-inflammatory  drug,  promotes  polyspermy  in  sea  urchins.  H. 
SCHUEL,  E.  TRAEGER,  R.  SCHUEL,  J.  BOLDT,  AND  M.  ALLIEGRO  (SUNY — 
Buffalo,  Buffalo,  NY). 

Sea  urchin  eggs  release  H2O2  during  the  cortical  reaction  at  fertilization  to  help  prevent  polypspermy 
by  inactivating  excess  sperm  near  the  egg  (Coburn  el  al.  1981,  Dev.  Biol.  84:  235-238;  Boldt  el  al.  1981, 
Gamete  Res.  4:  365-377).  This  process  resembles  the  peroxidatic  killing  of  bacteria  by  phagocytic  leu- 
kocytes during  inflammation.  Associated  with  these  reactions  in  leukocytes,  arachidonic  acid  is  oxidized 
via  the  cyclooxygenase  pathway  to  produce  prostaglandins  and  thromboxanes  as  well  as  oxygen-free 
radicals  and  H2O2.  Indomethacin  is  a  potent  inhibitor  of  cyclooxygenase  in  leukocytes.  Polyspermy  results 
when  Arbacia  punctualata  and  Strongylocentrotus  purpuralus  eggs  are  fertilized  in  10-100  \iM  indo- 
methacin.  The  incidence  of  polyspermy  depends  upon  the  concentration  of  indomethacin  and  the  number 
of  sperm  in  the  cultures.  Indomethacin  must  be  present  prior  to  completion  of  the  cortical  reaction  to 
promote  polyspermy.  Sperm  fertility  is  known  to  be  reduced  by  H2O2.  Indomethacin  does  not  protect 
sperm  from  inactivation  by  H2O2,  and  does  not  inhibit  the  sperm  peroxidase  that  uses  egg-derived  H2O2 
to  inactivate  sperm.  Indomethacin  apparently  acts  directly  on  the  eggs  to  promote  polyspermy.  Aspirin, 
which  is  a  less  potent  cyclooxygenase  inhibitor,  does  not  promote  polyspermy  at  5  mM  in  20  mM  Tris- 
buffered  sea  water  at  pH  8.0.  These  results  suggest  that  sea  urchin  eggs  may  oxidize  arachidonic  acid  by 
cyclooxygenase  to  help  assure  monospermic  fertilization. 

Supported  by  NSF  grant  #PCM-82-01561. 

A  study  of  the  heat  shock  response  in  early  embryos  o/Spisula  solidissima.  LAURIE 
E.  STEPHENS  (Physiology  Course,  MBL). 

Many  cell  types  and  organisms  exhibit  a  heat  shock  response  in  which  normal  cellular  protein 
synthesis  is  reduced  while  the  synthesis  of  a  new  set  of  proteins,  termed  the  heat  shock  proteins,  is 
induced.  I  have  performed  a  preliminary  characterization  of  the  heat  shock  response  in  embryos  of 
Spisula  solidissima.  This  was  done  by  following  in  vivo  protein  synthesis  with  35S-methionine  at  both 
normal  (2 1  °C)  and  elevated  temperatures.  Samples  were  run  on  1 5%  acrylamide  gels  and  autoradio- 
graphed.  The  heat  shock  response  is  most  clearly  observed  after  a  one  hour  exposure  to  3 1  °C,  although 
the  response  can  also  be  elicited  at  temperatures  ranging  from  29°  to  35°C.  Four  polypeptides  of  molecular 
weight  1 15,000,  72,000,  70,000,  and  37,000  daltons  appear  within  15  minutes  after  raising  the  temper- 
ature. Following  a  reduction  in  temperature  to  2 1  °C,  these  proteins  continue  to  be  synthesized  for  up 
to  four  hours,  although  normal  protein  synthesis  resumes  within  an  hour  under  these  conditions.  I  could 
detect  no  heat  shock  response  in  either  oocytes  or  embryos  prior  to  two  hours  after  fertilization. 

Heat  treatment  appears  to  initiate  de  novo  synthesis  of  the  mRNA  for  the  heat  shock  proteins.  Total 
RNA  was  extracted  from  embryos  and  assayed  in  the  reticulocyte  lysate  translational  system.  No  heat 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

shock  mRNA  was  detectable  in  control  embryos,  whereas  the  mRNA  from  heat-shocked  embryos  gave 
rise  to  heat-shocked  proteins  as  the  major  translation  products.  Interestingly,  however,  synthesis  of  most 
if  not  all  of  the  normal  proteins  was  also  specified  by  these  preparations,  although  their  synthesis  was 
barely  detectable  in  the  heat-shocked  embryos,  suggesting  the  existence  of  regulation  at  the  translational 
as  well  as  the  transcriptional  level. 

This  work  was  supported  by  NIH  training  grant  GM-3 11 36-04  to  the  Physiology  Course.  Thanks 
also  to  Tim  Hunt,  Eric  Rosenthal,  and  Andrew  Murray  for  invaluable  guidance  and  advice. 

A  low  molecular  weight  subunit  of  the  aggregation  factor  complex  of  Microciona 
prolifera  that  stoichiometrically  binds  to  and  inhibits  the  intact  aggregation  factor. 
PACHARA  VERAKALASA  AND  TOM  HUMPHREYS  (University  of  Hawaii). 

When  Ca++  is  removed,  the  20  million  dalton  aggregation  factor  (AF)  complex  of  M.  prolifera 
dissociates  into  subunits  which  inhibit  aggregation.  Inhibition  was  measured  by  defining  one  unit  as  the 
amount  required  to  suppress  aggregation  with  4  units  of  AF  for  30  min  at  22°C.  Four  units  of  AF  when 
dissociated  gave  one  unit  of  inhibition.  Ethanol  precipitation  and  permeation  chromatography  revealed 
ethanol-soluble  inhibitory  subunits  (IS)  of  less  than  10,000  daltons  which  contain  less  than  5%  of  the 
protein  and  polysaccharide  of  the  original  complex.  We  postulated  that  the  IS  is  a  monovalent  binding 
site  of  the  complex  which  binds  to  intact  AF  and  prevents  the  AF-AF  interaction  necessary  for  cell 
aggregation.  Such  binding  was  shown  by  mixing  AF  and  IS  and  passing  the  mixture  over  a  3000  A 
micropore  glass  bead  column  at  22°  in  10  2  or  10  3  M  Ca++.  On  this  column  AF  is  excluded  and  is 
separated  from  IS  which  is  fully  included.  When  64  units  of  AF  is  mixed  with  16  units  of  IS,  the  minimal 
amount  of  IS  required  to  inhibit  64  units  of  AF,  less  than  20%  of  the  AF  and  none  of  the  IS  is  recovered 
after  chromatography.  Apparently  the  two  components  remain  bound  to  each  other  and  do  not  separate 
on  the  column.  No  AF  or  IS  activity  is  recovered  from  a  mixture  of  64  units  of  AF  and  32  units  of  IS 
while  about  half  of  the  IS  activity  is  recovered  when  64  units  of  AFand  64  units  of  IS  are  chromatographed 
together.  The  binding  of  IS  to  AF  is  saturated  at  a  ratio  of  one  unit  IS  to  two  units  AF.  At  10"4  A/ Ca++ 
binding  did  not  occur  and  both  AF  and  IS  are  fully  recovered  after  chromatography  of  a  mixture. 

Tissue-specific  expression  of  tubulin  RNAs  during  sea  urchin  development.  KRISTI 
WHARTON,  GLENN  MERLINO,  RUDOLF  RAFF,  AND  JOAN  RUDERMAN  (Marine 
Biological  Laboratory). 

We  have  examined  tubulin  gene  expression  in  sea  urchin  embryonic  ectoderm  and  endoderm. 
Ectodermal  cells  were  dissociated  from  Lytechinus  pictus  plutei  by  treatment  with  an  isotonic  glycine- 
EDTA  solution  and  purified  by  filtration  through  a  28  nm  Nitex  mesh.  Preparations  of  endodermal 
tissues  were  collected  from  Triton  X-100  treated  plutei  by  differential  centrifugation.  RNA  was  isolated 
from  each  tissue  preparation  by  a  guanidine-HCl  extraction,  electrophoresed  on  agarose  gels,  and  trans- 
ferred to  nitrocellulose  filters. 

Lytechinus  pictus  cDNA  clones  complementary  to  a-tubulin  (p«2)  and  to  /3-tubulin  (p/32)  sequences 
were  used  as  hybridization  probes  for  tubulin  mRNAs.  Total  cellular  pluteus  RNA  contains  two  /3-tubulin 
RNA  transcripts  of  1.8  and  2.2  kb  in  length,  which  probably  represent  mature  mRNAs,  as  well  as  more 
weakly  hybridizing  bands  of  4.5,  6.5,  and  15  kb  in  size.  The  «-tubulin  message  is  1.75  kb  in  length,  and 
weaker  high  molecular  weight  RNAs  were  found  at  2.3,  2.6.  3.8,  4.5,  and  15  kb.  The  levels  of  both  «- 
and  0-tublin  mRNAs  are  considerably  higher  in  ectodermal  than  in  endodermal  cells  (2-5  fold). 

The  ectodermal  cells  of  intact  sea  urchin  embryos  may  be  deciliated  by  a  hypotonic  seawater  shock. 
Plutei  were  treated  by  three  rounds  of  deciliation,  each  followed  by  a  90  min  recovery  time.  These 
deciliated  embryos  exhibited  a  4-6  fold  increase  in  mature  tubulin  message  sequences  (fi-,  1.8  and  2.2 
kb;  a-,  1.75  kb)  in  the  ectoderm.  In  contrast  the  endodermal  cells  showed  at  most  a  1.5  fold  increase  in 
tubulin  sequences.  The  high  molecular  weight  band  sequences  show  a  3-4  fold  increase  in  response  to 
deciliation.  These  larger  sequences  may  be  nuclear  precursors  since  they  are  readily  detected  in 
nuclear  RNA. 

The  high  levels  of  tubulin  mRNAs  in  ectoderm  are  consistent  with  the  heavy  ciliation  of  the  ectoderm 
and  with  the  higher  le\el  of  tubulin  proteins  observed  by  2-D  gel  analysis  of  pluteus  tissue.  As  in  other 
systems  (e.g.  Chlamydomonas  and  Tetrahymena)  the  response  of  ectodermal  cells  to  deciliation  requires 
augmented  synthesis  of  tubulin  mRNAs.  Both  /J-tubulin  mRNAs  increase  in  deciliated  embryos  suggesting 
that  either  both  encode  ciliary  tubulin  or  that  deciliation  induces  a  non-specific  rise  in  tubulin  synthesis. 

This  work  was  supported  by  NIH  grant  #HD15351  (to  J.V.R.)  and  the  Embryology  course  at  MBL. 


FERTILIZATION  AND  DEVELOPMENT  379 

Lucifer  yellow  CH  as  a  non-intrusive,  in  vivo  fluorescent  probe  for  physiological 
studies  during  early  development.  R.  I.  WOODRUFF,  D.  A.  LUTZ,  AND  S.  INOUE 
(Marine  Biological  Laboratory). 

We  used  the  fluorescent  dye  Lucifer  yellow  CH  for  following  early  developmental  events  directly 
in  living  embryos.  This  negatively  charged  dye  is  freely  diffusable  through  gap  junctions  for  ca.  30  minutes, 
after  which  it  binds  to  cell  constituents;  its  fluoresence  is  proportional  to  concentration  (Stewart  1978, 
Cell  14:  741-759). 

We  monitored  distribution  of  iontophoretically  micro-injected  dye  by  a  high  sensitivity  (SIT)  video 
camera  attached  to  a  microscope  with  crossed  polarizers  and  appropriate  niters  in  the  trans-illumination 
mode.  The  fluorescent  image  of  dye-injected  sea  urchin  blastomeres  was  too  faint  to  be  detected  by  the 
dark-adapted  eye  but  was  clearly  displayed  through  the  SIT  camera.  With  a  video  analyzer,  we  could 
monitor  the  rising  level  of  fluorescence  injection  and  graphically  display  the  intensity  distribution  of  the 
diffusing  dye,  sharply  peaked  at  the  tip  of  the  injection  needle.  The  volcano-shaped  distribution  converted 
to  an  ellipse  conforming  to  the  cell  shape  within  seconds,  once  the  injecting  current  was  turned  off.  The 
amount  of  dye  rose  linearly  during  iontophoresis,  became  constant  thereafter,  and  was  unaffected  by 
change  in  cell  shape  during  cleavage. 

Individual  micromeres  were  injected  with  Lucifer  yellow  at  the  16-cell  stage  and  their  development 
followed.  Scattered  throughout  the  rings  of  primary  mesenchyme  cells,  about  one-quarter  of  the  cells 
displayed  Lucifer  fluorescence;  in  contrast  a  quadrant  of  the  archenteron  also  fluoresced  at  the  gastrula 
stage.  At  the  prism  and  pluteus  stages,  fluorescently  labeled  offspring  of  the  single  injected  micromere 
were  seen  crawling  along  the  birefringent  spicules.  The  cells  divided  and  developed  normally  and  syn- 
chronously with  non-injected  sister  cells. 

This  non-intrusive  technique  allows  one  to  trace  the  fate  of  individual  cells  with  great  precision  and 
shows  promise  for  quantitating  the  fluorescence  in  localized  regions  within  living  cells. 

Supported  by  grants  NSF  PCM  77-16455  (R.I.W.),  NSERC  post-graduate  fellowship  (D.A.L.),  NSF 
PCM  79-22136,  and  NIH  7R01-GM  31617  (S.I.). 

NEUROBIOLOGY 

Trigonometric  nearest  neighbor  analysis  of  the  neuroplasmic  lattice  arrays  in  axons. 
W.  J.  ADELMAN,  JR.  (Laboratory  of  Biophysics,  NINCDS,  MBL),  A.  J.  HODGE, 
AND  R.  B.  WALTZ. 

Electron  micrographs  of  transverse  sections  of  myelinated  axons  found  in  sciatic  nerves  of  the  toad, 
Bufo  woodhousi  fowlerii,  were  examined  so  as  to  measure  the  spatial  characteristics  of  the  lattice  array 
of  filamentous  elements  in  the  axoplasm.  Cross-sections  through  the  Schmidt-Lanterman  "cleft"  regions 
were  chosen  for  analysis  because  the  axons  were  invariably  constricted  in  these  zones,  with  a  consequently 
higher  packing  density  of  longitudinal  filamentous  elements  and  seemingly  better  preservation  of  order 
than  that  found  in  internodal  regions.  The  analytical  technique  involved  use  of  a  TV  camera  to  produce 
a  monochrome  image  of  a  print  on  a  color  terminal  linked  to  a  PDP-11/60  computer.  Appropriate 
programming  allowed  the  cursor  coordinates  to  be  inserted  into  a  memory  file  upon  command  following 
placing  of  the  cursor  over  the  observed  lattice  locations  of  the  neurofilaments  and/or  neurotubules. 
Insertion  of  coordinates  into  memory  was  confirmed  by  an  "echo  subroutine"  which  generated  a  bright 
spot  on  the  screen  at  the  cursor  position,  thus  eliminating  duplication  of  digitized  points  and  allowing 
direct  visualization  of  the  matrix  in  memory.  A  subroutine  was  written  to  analyze  the  data.  For  each 
memory  location,  the  routine  searched  for  all  other  locations  within  a  specified  vector  radius,  and  thereby 
generated  (a)  the  vector  length  distribution,  (b)  the  number  of  neighbors  for  each  point,  and  (c)  the  angles 
between  vectors  to  nearest  neighbors.  The  program  was  tested  and  proven  operational  using  an  electron 
micrograph  of  a  transverse  section  of  blow  fly  flight  muscle  in  which  the  myofilaments  were  located  in 
a  known  hexagonal  array.  Analysis  of  the  neuroplasmic  lattice  in  toad  axons  showed  a  relative  invariance 
of  the  angular  distribution  with  increasing  vector  length  as  compared  with  the  upward  progression  of 
nearest  neighbor  number.  This  analysis  indicated  that  the  lattice  most  clearly  approximates  a 
hexagonal  array. 

Fast  axonal  transport  in  lobster  axons.  ROBERT  D.  ALLEN  (Dartmouth  College), 
RAYMOND  J.  LASEK,  SUSAN  P.  GILBERT,  ALAN  J.  HODGE,  AND  C.  K.  GOVIND. 

The  motor  axons  to  the  claw  closer  muscle  of  juvenile  lobsters,  Homarus  americanus,  are  valuable 
for  examining  fast  axonal  transport  since  they  contain  microtubules  but  not  neurofilaments.  Using  the 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Allen  video  enhanced  contrast  (AVEC)  system  with  polarized  light  (POL)  microscopy  or  differential 
interference  contrast  (DIC)  microscopy,  rapid  participate  movements  can  be  seen  in  a  field  of  21  ^m 
along  any  selected  region  of  the  axon.  Three  size  categories  of  organelles  are  observed  to  move  in  both 
the  orthograde  and  retrograde  directions.  Mitochondria  2-20  nm  (or  more)  in  length  undergo  interrupted 
movements  in  either  direction.  Some  of  these  movements  appear  to  be  elastic  recoil.  They  also  perform 
"acrobatic  maneuvers,"  such  as  "loop-the-loop"  and  "snake  descending  a  staircase"  movements.  Medium 
size  (0.35-1.0  ^m)  particles,  thought  to  be  mostly  vesicular  elements,  exhibit  discontinuous  movements, 
and  their  rates  of  movement  are  more  rapid.  Much  greater  numbers  of  small  particles  (<0.2  ^m)  the  size 
of  synaptic  vesicles  and  other  tubulovesicular  elements  move  in  the  orthograde  direction  at  an  average 
velocity  of  3.84  ±  0.88  nm.  Fewer  particles  in  this  size  range  move  in  the  retrograde  direction  slightly 
more  rapidly  (4.18  ±  0.95  fim/sec).  In  most  but  not  all  instances,  organelles  and  small  particles  can  be 
seen  to  move  along  longitudinally  oriented  linear  elements  which  are  presumably  microtubules  since 
micrographs  of  those  same  axons  show  a  network  of  microtubules  with  few  if  any  neurofilaments.  The 
apparently  smooth,  continuous  movement  of  small  particles  in  either  the  orthograde  or  retrograde  di- 
rection is  believed  to  be  the  fundamental  process  of  fast  axonal  transport  because  these  particles,  unlike 
the  larger  organelles,  are  programmed  to  move  in  a  single  direction.  At  the  light  microscope  level  it  is 
clear  that  these  movements  are  seen  in  the  vicinity  of  microtubules.  In  the  lobster,  the  microtubular- 
based  system  seems  adequate  to  support  fast  axonal  transport  without  invoking  a  possible  role  for  neu- 
rofilaments. 

Supported  in  part  by  an  NIH  grant,  GM  27284  to  R.D.A. 

Seasonal  changes  in  the  dread i an  modulation  of  sensitivity  of  the  Limulus  lateral 
eye.  ROBERT  B.  BARLOW  JR. 

A  circadian  clock  in  the  Limulus  brain  generates  efferent  optic  nerve  activity  at  night  (Science  197: 
86-89,  1977).  The  efferent  activity  changes  the  structure  and  function  of  the  lateral  compound  eyes 
causing  a  dramatic  increase  in  retinal  sensitivity  at  night  (Science  210:  1037-1039,  1980). 

I  report  here  that  the  circadian  modulation  of  retinal  sensitivity  changes  with  the  time  of  year. 
During  the  summer,  circadian  rhythms  in  the  amplitude  of  the  ERG  exhibit  short  nights  relative  to  those 
measured  during  the  winter.  In  both  cases  animals  were  exposed  several  weeks  to  the  natural  light-dark 
cycle  of  sunlight  and  then  clamped  to  a  rigid  platform  in  an  aquarium  located  in  a  light  proof,  shielded 
cage.  ERG's  elicited  by  dim,  brief  flashes  presented  every  1 5  minutes  were  recorded  with  corneal  electrodes 
while  the  animals  remained  in  the  dark.  The  amplitudes  of  the  ERG's  were  plotted  and  measurements 
were  made  of  the  animals  "subjective  night  length",  which  is  defined  as  the  number  of  hours  the  ERG 
amplitude  exceeds  25%  of  the  difference  between  the  daytime  and  nighttime  levels.  Measurements  from 
75  Limuli  yielded  subjective  night  lengths  ranging  from  9  to  1 1  h  in  the  summer  and  from  13  to  15  h 
in  the  winter.  Intermediate  values  were  recorded  in  the  spring  and  fall.  The  seasonal  changes  in  visual 
responses  corresponded  reasonably  well  to  the  seasonal  changes  in  sunset  and  sunrise  for  43°N  latitude. 
No  seasonal  changes  were  detected  in  the  circadian  periods. 

The  circadian  clock  appears  to  adapt  its  duty  cycle  for  generating  efferent  activity  to  the  seasonal 
changes  in  night  length.  Do  the  seasonal  changes  reflect  continuous  adjustments  of  the  circadian  clock 
to  fluctuations  in  daylight?  Or  are  they  generated  by  an  endogenous  circannual  clock? 

I  thank  Joseph  Fladd  for  technical  assistance.  Supported  by  NIH  grant  EY-00667  and  NSF  grant 
BNS  81-19436. 

Somatotopy  within  the  medullary  electrosensory  nucleus  of  the  skate,  Raja  erinacea. 
DAVID  BODZNICK  (Wesleyan  Univ.,  Middletown,  CT)  AND  ANNE  W.  SCHMIDT. 

Ampullae  of  Lorenzini  are  electrosensory  organs  innervated  by  the  anterior  lateral  line  nerve  (ALLN) 
on  the  head  and  pectoral  fins  of  elasmobranchs.  Anatomical  (Koester  and  Boord  1978,  Am.  Zool.  17: 
431)  and  physiological  studies  (Bodznick  and  Northcutt  1980,  Brain  Res.  195:  313)  have  demonstrated 
that  electroreceptor  afferents  terminate  in  the  medullary  dorsal  nucleus  (DN).  We  now  report  that  the 
terminations  of  these  electroreceptive  fibers  are  somatotopically  organized. 

The  electrosensor-  organs  of  skates  occur  in  three  major  groups  on  each  side  of  the  body,  innervated 
by  three  separate  ALL  -ami.  In  7  animals  the  proximal  cut  end  of  an  individual  ramus  was  soaked  in 
1%  lysolecithin  nearly  saturated  with  HRP.  After  8-16  days  the  animals  were  perfused  with  glutaraldehyde 
and  brain  sections  examined  for  peroxidase  activity  (TMB  reaction). 

The  three  ALLN  rami  innervating  clusters  of  ampullae  project  to  non-overlapping  portions  of  the 
DN  neuropil.  The  external  mandibular  ramus  that  innervates  the  largest  and  most  caudal  hyoid  cluster 
of  ampullae  terminates  in  a  large  dorsal  portion  of  DN.  The  superficial  ophthalmic  ramus  from  the  most 
rostral  ampullae  on  the  snout  projects  to  the  most  ventral  portion  of  DN,  and  the  buccal  ramus  innervating 


NEUROBIOLOGY  381 

ampullae  on  the  lateral  part  of  the  head  terminates  in  the  central  portion.  These  dorso-ventral  divisions 
can  be  recognized  in  normal  nissl-stained  sections  as  distinct  areas  separated  by  compact  cell  plates. 

In  single-cell  recordings  the  receptive  field  maps  of  DN  neurons  confirmed  this  organization.  In- 
dividual electrode  tracks  revealed  that  the  most  dorsal  cells  received  their  input  from  small  numbers  of 
ampullae  of  the  most-caudal  hyoid  group  and  cells  with  buccal  or  superficial  opthalmic  inputs  were 
encountered  ventrally  in  DN. 

The  wide  distribution  of  ampullary  organs  on  the  body  surface  of  skates  provides  a  means  of 
localizing  electric  field  sources  (e.g.  prey  animals).  This  spatial  information  is  preserved  within  the  med- 
ullary electrosensory  nucleus. 

This  work  was  made  possible  by  The  Grass  Foundation  and  an  NIH  grant  to  D.B. 

Fast  axonal  transport  in  isolated  axoplasm  of  Myxicola  infundibulum.  ANTHONY 
C.  BREUER  (Cleveland  Clinic  Foundation),  PETER  A.  M.  EAGLES,  SUSAN  P. 
GILBERT,  ROBERT  D.  ALLEN,  JANIS  METUZUALS,  DAVID  F.  CLAPIN,  AND 
ROGER  D.  SLOBODA. 

The  giant  axon  of  the  marine  fan  worm,  Myxicola  infundibulum,  has  received  considerable  attention 
because  of  the  unusual  preponderance  of  neurofilaments  and  paucity  of  microtubules  in  the  axoplasmic 
cytoskeleton  and  the  ready  accessibility  of  the  axoplasm,  which  can  be  pulled  out  of  the  giant  axon  from 
the  intact  organism  in  10  seconds  (Gilbert  1972,  Nature  New  Biol.  237:  195-197).  We  report  the  visu- 
alization of  moving  organelles  in  Myxicola  axoplasm  using  AVEC-DIC  video-enhanced  microscopy 
(Allen  el  al.  1981,  Cell  Mot  Hit  y  1:  291-302),  a  Hamamatsu  C-1000  Chalnicon  camera  and  Polyprocessor 
frame  memory  to  subtract  out-of-focus  background  mottle.  Specimens  collected  in  England  were  rinsed 
in  calcium-free  sea  water  and  axoplasm  was  removed,  sandwiched  between  two  No.  0  coverglasses, 
surrounded  by  1 .01  osmolal  glutamate  buffer  pH  7.0  with  0.5  mAf  ATP  and  immediately  examined  with 
the  microscope.  Translocation  of  mitochondria,  intermediate  sized  particles  (about  200-300  nm  diam- 
eter), and  small  particles  (about  <100  nm  diameter)  was  readily  visualized  and  persisted  for  up  to  2 
hours.  Transport  was  bidirectional  for  all  classes  of  particles  and  could  be  seen  throughout  the  axoplasm, 
although  the  preponderance  of  traffic  was  noted  near  the  surface.  Distinct  linear  organelles  could  be  seen 
in  some  sequences,  and  elongate  mitochondria  and  smaller  organelles  could  be  seen  moving  along  them, 
at  times  retracing  their  progress  for  more  than  20  nm  along  the  same  "track."  We  interpret  these  motions 
as  rapid  axonal  transport  of  membranous  organelles  along  linear  elements  believed  to  be  microtubules. 
Ultrastructural  analyses  are  in  progress.  The  small  numbers  of  distinctly  visible  and  spatially  separate 
linear  elements  seen  in  the  axoplasm  by  AVEC-DIC  microscopy  and  the  vastly  fewer  translocating 
organelles  relative  to  squid  and  lobster  axons  may  make  this  system  simpler  to  analyze.  Further  study 
of  isolated  axoplasm  of  the  Myxicola  giant  axon  may  prove  useful  in  unraveling  the  molecular  mechanism 
of  fast  nerve  cell  transport. 

A  relatively  robust,  single-trial,  associative  learning  in  the  opisthobranch  mollusc, 
Pleurobranchaea  californica.  L.  B.  COHEN  AND  J.  E.  FREEDMAN  (Dept.  of  Phys- 
iology, Yale  University  School  of  Medicine). 

With  the  aim  of  developing  a  preparation  with  relatively  few,  large  neurons  that  could  be  used  for 
studies  of  the  cellular  basis  of  learning,  we  have  carried  out  behavior  experiments  on  Pleurobranchaea. 
The  paradigm  we  have  used  is  called  taste-aversion  learning,  a  subset  of  the  paradigms  called  classical 
conditioning. 

For  each  experiment  5-8  animals  were  divided  into  two  groups.  Both  groups  were  tested  to  determine 
the  concentrations  of  lobster  extract  and  honey  necessary  to  elicit  a  criterion  response  (partially  everted 
proboscis).  One  to  four  hours  later  one  of  the  foods  was  paired  with  a  1-3  rng/ml  solution  of  quinine, 
an  aversive  substance.  After  a  further  delay  of  1  to  24  hours,  the  animals  were  retested  to  see  if  the 
quinine-pairing  affected  the  concentration  of  food-substance  needed  to  elicit  the  criterion  response.  In 
our  best  experiment,  the  pairing  led  to  a  relative  decrease  in  response  to  the  paired  food  when  compared 
to  the  control  food  in  each  animal.  Statistical  analysis  of  the  results  from  this  experiment  showed  that 
the  results  could  occur  by  chance  with  a  probability  of  less  than  0.02.  Six  additional  experiments  of  this 
kind  were  done.  When  the  results  of  all  seven  were  combined,  the  mean  change  was  in  the  expected 
direction  and  the  probability  that  the  result  was  due  to  chance  was  less  than  0.00 1 .  The  mean  relative 
increase  in  concentration  of  the  paired  taste  needed  to  elicit  the  criterion  response  was  a  factor  of  3. 

While  we  are  hopeful  that  changes  in  the  protocol  can  lead  to  an  even  more  robust  and  larger 
behavioral  change,  we  think  that  the  results  already  obtained  are  good  enough  to  allow  us  to  begin  cellular 
studies. 

Supported  by  N.I.H.  grant  NS  08437. 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Pharmacological  properties  of  isolated  and  cultured  horizontal  cells  of  the  skate 
retina.  JOHN  E.  DOWLING  (Harvard  University),  ERIC  M.  LASATER,  AND  HARRIS 
RIPPS. 

The  ability  to  maintain  intact,  identifiable  nerve  cells  in  culture  affords  a  unique  opportunity  to 
study  the  interaction  of  neurotransmitter  candidates  with  cell-surface  receptors.  In  the  present  study, 
horizontal  cells  were  isolated  from  the  all-rod  retina  of  the  skate  by  treating  the  retina  with  papain  in 
Leibovitz's  tissue  culture  medium  (L-15),  adjusted  for  isotonicity  with  skate  CSF.  The  cells  were  me- 
chanically disassociated  by  repeated  pipetting,  plated  out  in  tissue  culture  dishes  containing  the  modified 
medium,  and  maintained  in  culture  for  up  to  three  weeks.  Although  the  horizontal  cells  tended  to  alter 
their  shapes  and  to  retract  their  fine  processes  during  the  first  24  h  in  culture,  they  retained  most  of  their 
morphological  features  and  began  to  sprout  new  processes  during  the  culture  period.  Prior  to  determining 
the  pharmacological  properties  of  the  cells,  the  culture  medium  was  replaced  by  an  elasmobranch  Ringer's 
solution. 

The  results  reported  here  were  obtained  by  intracellular  recording  from  cells  maintained  in  culture 
for  2-5  days.  Immediately  after  penetration,  horizontal  cells  had  resting  membrane  potentials  of  -15 
mV  to  -30  mV,  and  input  resistances  of  50-70  megohm.  Within  a  few  minutes,  however,  resting 
potentials  usually  increased  to  final  values  of  between  -70  and  -90  mV,  and  input  resistances  reached 
150  megohm.  Cells  with  resting  potentials  greater  than  -60  mV  were  tested  for  their  responsiveness  to 
transmitter  agents  applied  via  pressure  ejection  through  multi-barreled  pipettes.  L-glutamate  and  the 
glutamate  analogs  quisqualate  and  kainate  produced  depolarizations  of  up  to  90  mV  at  concentrations 
(in  the  delivery  pipettes)  of  less  than  100  \iM.  No  responses  to  L-aspartate  were  observed  unless  5  mM 
or  more  of  drug  was  used.  The  cells  were  also  highly  responsive  to  -y-aminobutyric  acid  (GABA);  con- 
centrations of  less  than  100  \tM  GABA  produced  long-lasting  depolarizations  of  up  to  80  mV  that 
resembled  the  glutamate  responses.  The  responses  to  GABA  could  be  partially  blocked  by  bicuculline. 
Skate  horizontal  cells  in  culture  were  unresponsive  to  D-glutamate,  glycine.  D-  and  L-aspartate,  dopamine, 
carbachol,  and  serotonin  applied  at  concentrations  of  1  mM. 

This  research  was  supported  by  grants  EY  00824  and  EY  00285  from  the  National  Eye 
Institute,  USPHS. 

Circadian  clock  generates  efferent  optic  nerve  activity  in  the  excised  Limulus  brain. 
LESLIE  E.  EISELE,  LEONARD  KASS,  AND  ROBERT  B.  BARLOW,  JR.  (Syracuse 

University,  NY). 

A  circadian  clock  in  Limulus  brain  generates  efferent  optic  nerve  activity  at  night  leading  to  various 
changes  in  retinal  structure  and  function  (Science  197:  86-89,  1977;  Science  210:  1037-1039,  1980).  We 
developed  an  excised  brain  preparation  to  study  efferent  and  afferent  connections  to  this  circadian  clock. 
The  brain  was  dissected  free  from  the  rest  of  the  animal  and  placed  into  a  temperature-controlled  chamber 
filled  with  an  organ  culture.  Glass  suction  electrodes  were  positioned  along  the  various  desheathed  optic 
nerve  stumps.  Occasionally  we  recorded  from  or  electrically  stimulated  different  bundles  of  the 
same  nerve. 

Efferent  activity  recorded  from  the  lateral  optic  nerve  (LON)  in  situ  resembles  that  recorded  from 
the  excised  brain  in  the  following  ways:  the  efferent  activity  which  begins  in  the  early  evening  occurs  in 
discrete  bursts,  and  the  general  level  of  activity  changes  from  day  to  night.  Efferent  activity  persists  for 
up  to  3  days  in  the  excised  brain.  The  bursting  efferent  activity  recorded  from  an  LON  is  synchronous 
with  that  recorded  from  the  opposite  LON,  the  median  optic  nerve  (MON),  and  the  ventral  eye  nerve 
(YEN).  Bisecting  the  isolated  protocerebrum  desynchronizes  the  bursts  of  efferent  activity  in  opposite 
LONs.  Thus,  efferent  cell  bodies  are  located  in  both  sides  of  the  protocerebrum.  Further  lesions  of  the 
brain  suggest  that  the  location  of  the  cell  bodies  may  be  limited  to  the  lamina  or  medulla. 

Electrically  stimulating  both  MONs  induces  efferent  activity  recorded  from  both  LONs.  Illuminating 
the  excised  brain  tends  to  inhibit  the  efferent  activity  in  LON. 

In  sum,  the  excised  brain  appears  to  be  a  viable  preparation  for  further  studies  on  central  visual 
pathways  in  Limulus. 

Supported  by  N1H    ^ants  EY-00667  and  EY-05443  and  NSF  grant  BNS  81-19436. 

EM  and  A  VEC-DIC  analyses  of  membranous  organelle  transport  in  squid  giant 
axons  and  isolated  axoplasm.  M.  A.  FAHIM,  S.  T.  BRADY,  A.  HODGE,  AND 
R.  J.  LASER  (Anatomy  Dept.,  Case  Western  Reserve  Univ.,  Cleveland,  OH). 

Recent  developments  in  video  enhanced  light  microscopy  (A VEC-DIC)  permit  visualization  of 
particles  moving  in  both  orthograde  and  retrograde  directions  in  the  squid  giant  axon  and  isolated 


NEUROBIOLOGY  383 

axoplasm.  The  dominant  feature  in  these  studies  is  the  presence  of  vast  numbers  of  small  particles  and 
other  tubulovesicular  elements  moving  parallel  to  linear  elements  in  both  directions.  In  order  to  analyze 
these  particles  and  relate  them  to  identified  cellular  organelles,  axonal  transport  was  blocked  focally  by 
cooling  a  3  mm  region  of  the  axon  to  4°C  for  1-4  h.  Many  more  moving  particles  were  observed  adjacent 
to  the  cold  block  area.  On  the  proximal  side  of  the  cold  block  large  numbers  of  small  particles  and  other 
tubulovesicular  elements  were  most  frequently  seen  moving  parallel  to  linear  elements.  By  contrast, 
medium  size  vesicles  and  large  membranous  bodies  were  enriched  on  the  retrograde  side  of  the  block. 
Similar  results  were  obtained  from  intact  axons  and  isolated  axoplasm. 

Using  4%  glutaraldehyde  in  EGTA-phosphate  buffer  ( 1 200  mosm,  pH  7.2),  intact  axons  and  extruded 
axoplasm  were  fixed  and  prepared  for  EM.  After  locally  cooling  the  extruded  axoplasm  orthogradely 
transported  particles  accumulated  just  proximal  to  the  cooled  site  resulting  in  a  distinct  increase  in  the 
number  of  small  particles  at  that  site.  Electron  micrographs  revealed  that  the  small  particles  are  mostly 
tubular  and  vesicular  structures  (40-50  nm  in  diameter)  which  accumulated  in  files  parallel  to  the  long 
axis  of  the  axon.  Many  of  the  small  vesicles  were  similar  in  size  to  synaptic  vesicles.  The  particles 
accumulating  in  the  retrograde  direction  tended  to  be  larger  (80-100  nm)  and  included  many  double 
membrane  structures.  Particles  accumulating  in  both  directions  have  a  dense  granular  material  associated 
with  the  pathways.  Microtubules  were  less  frequent  in  the  cooled  area,  while  neurofilaments  were  ap- 
parently unaffected  by  the  cold.  These  results  suggest  that  different  identifiable  axonal  components  travel 
in  different  directions  along  the  axon  in  association  with  linear  pathways. 

Supported  by  a  Grass  Fellowship  to  Dr.  M.  A.  Fahim. 

Membrane  changes  in  a  single  photoreceptor  cause  retained  associative  behavioral 
changes  in  Hermissenda.  JOSEPH  FARLEY,  WILLIAM  G.  RICHARDS,  LORRAINE 
LING,  EMILY  LIMAN,  AND  DANIEL  L.  ALKON  (Section  on  Neural  Systems,  Lab. 
of  Biophysics,  NINCDS,  NIH,  MBL). 

Previous  research  with  Hermissenda  has  demonstrated  striking  correlations  between  the  associative 
suppression  of  phototaxis  and  biophysical  changes  intrinsic  to  two  of  the  three  type  B  photoreceptors. 
Repeated  light-rotation  pairings  produce  cumulative  depolarization  and  probable  increase  in  intracellular 
Ca++  in  the  type  B  cells.  This  results  in  long-term  inactivation  of  a  fast,  outward  K+  current  (IA)  in  B 
cells,  observable  for  days  following  training.  We  now  report  that  type  B  cells  are  causally  related  to 
associative  modification  of  phototaxis. 

Single  type  B  photoreceptors  were  impaled  in  restrained  animals,  and  were  then  exposed  to  either: 
1)  five  pairings  (at  2-min  intervals)  of  30  sec  of  light  and  depolarizing  (+15  mV)  current,  or  2)  five 
unpaired  (i.e.  separated  by  30  sec)  presentations  of  light  and  current  (at  2-min  intervals).  For  a  third 
"sham"  treatment,  intracellular  penetration  of  B  cells  lasted  for  less  than  5  min.  Measurements  of  changes 
in  membrane  potential  and  resistance  were  obtained  5  min  following  training.  Animals  were  then  allowed 
to  recover  and  were  subsequently  re-tested  for  phototaxis. 

Light-current  pairings  produced  a  cumulative  depolarization  of  5.23  mV  (±S.E.M.  of  0.79  mV)  in 
B  cells  (n  =  24),  which  was  absent  for  cells  exposed  to  the  unpaired  treatment  (AVm  =  0.83  mV  ±  0.93; 
/(45)  =  3.55,  P  <  0.001).  Input  resistance  was  also  increased  for  the  paired  (31.60  Mfi  ±  1.37  to  47.10 
MSi  ±  2.25;  t  =  2.05,  P  <  0.05)  but  not  unpaired  (38.53  M12  ±  1.13  to  39.65  MO  ±  1.69)  treatment 
conditions,  by  48%. 

For  those  animals  which  recovered,  "blind"  measurement  of  phototaxic  latencies  48  h  post-training 
revealed  a  pairing-specific  suppression  of  phototaxis.  Test  latencies  were  significantly  longer  for  paired 
(102.00  min  ±  7.27;  n  =  13)  vs.  unpaired  (52.65  min  ±  21.76;  n  =  6;  r(17)  =  2.56;  P  <  0.01)  and  vs. 
sham  (67.60  min  ±  18.03;  n  =  7;  /(1 8)  =  1.98,  p  <  0.05)  treatments,  which  did  not  differ. 

Kits  of  voltage-sensitive  fluorescent  probes  for  external  or  iontophoretic  staining  of 
central  nervous  systems  or  single  neurons.  A.  GRINVALD  (Weizmann  Institute 
of  Science),  R.  HILDESHEIM,  J.  PINE,  AND  L.  B.  COHEN. 

Recent  experiments  on  several  different  preparations  indicated  that  some  of  the  best  probes  evaluated 
on  squid  giant  axons  are  not  useful  for  optical  monitoring  of  neuronal  activity  in  other  preparations. 
However,  in  such  cases,  close  analogs  were  often  found  to  give  large  signals.  Therefore  we  have  synthesized 
45  analogs  of  styryl  dyes.  We  designed  families  of  probes  whose  net  charges  are  either  negative,  neutral, 
positive,  or  doubly  positive.  The  length  of  the  conjugated  chain  was  also  varied  (two,  four,  or  six  carbons). 
The  aliphatic  substituents  on  the  anilino  nitrogen  were  varied  from  a  methyl  to  a  hexyl.  All  of  these  dyes 
were  tested  on  squid  giant  axons,  in  voltage-clamp  experiments.  The  largest  fluorescence  signals  were 
obtained  with  analogs  having  the  dipenthyl-anilino  chromophore.  However,  when  these  dyes  were  tested 
on  other  preparations  they  were  not  uniformly  successful.  Even  though  RH-42 1 ,  the  dipentylanilino  and 


384  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

sulfobutyl  styryl,  exhibited  a  fractional  change  of  25%/100  mV  when  tested  on  neuroblastoma  cells 
maintained  in  culture,  in  experiments  on  Aplysia  neurons  maintained  in  culture  this  dye  gave  small 
signals.  For  Aplysia,  RH-376  (the  propyl  phosphonate  analog  of  RH-160)  had  to  be  used  to  obtain  large 
signals. 

To  allow  optical  measurements  of  synaptic  responses  from  the  site  of  synapses  on  dendrites,  we  have 
designed  the  doubly  positively  charged  dyes  for  iontophoretic  injection  into  single  cells.  We  found  that 
dyes  with  short  alkyl  groups  (RH-355  and  RH-461)  on  the  anilino  nitrogen  diffused  quickly  into  the 
processes  of  injected  leech  neurons.  These  dyes  are  the  dimethyl  and  diethyl  analogs  and  have  a  tri- 
methylammonium  propyl  side  chain  and  four  carbons  in  the  conjugated  chain. 

For  optical  recording  of  cortical  activity  in  the  mammalian  brain,  many  of  these  dyes  were  evaluated 
by  testing  them  on  the  rat  visual  cortex,  and  again  many  did  not  perform  well.  However,  RH-292  (a 
triethyl  ammonium  propyl.  dibutyl  anilino  styryl)  did  (see  Orbach  el  at.  1982,  Biol.  Bull.  163:  389). 

We  conclude  that  optical  monitoring  of  membrane  potential  is  more  likely  to  succeed  if  10  to  30 
voltage  sensitive  dyes  rather  than  a  few  probes  can  be  evaluated  for  each  given  preparation. 

Supported  by  a  grant  from  the  U.  S.  Israel  Binational  Science  Foundation  and  an  NIH  grant 
NS08437. 

Central  organization  of  vest  ibid  ar  efferent  neurons  in  the  toad  fish,  Opsanus  tau. 
STEPHEN  M.  HIGHSTEIN  AND  ROBERT  BAKER  (Marine  Biological  Laboratory). 

The  semicircular  canals,  saccule,  and  lateral  line  organs  are  innervated  by  efferent  vestibular  neurons 
whose  cell  bodies  lie  in  a  medial  nucleus  in  the  medulla  below  the  cerebellum.  Efferent  somata  were 
distributed  rosto-caudally  between  two  superficial  dorsal  commissures  200  j/m  apart.  Coronally  they  lay 
in  a  dorsal  subgroup  above  and  a  ventral  subgroup  along  the  median  longitudinal  fasiculi.  Saccular  and 
lateral  line  efferents  overlapped  those  of  the  canals  with  an  additional  10%  located  200  ^m  behind  the 
more  caudal  commissure.  Saccular  efferents  comprised  most  of  the  dorsal  subgroup  and  canal  efferents 
the  ventral.  However,  a  single  neuron  efferent  to  any  end  organ  could  be  in  either  subdivision.  Efferent 
neurons  were  always  found  bilaterally  but  with  an  ipsilateral  predominance  (3  to  1).  Each  semicircular 
canal  was  innervated  by  30-40  neurons;  saccular  efferents  numbered  140-150.  Dendrites  of  efferent 
neurons  from  the  dorsal  subgroup  interdigitated  bilaterally,  those  located  ventrally  were  exclusively  ip- 
silateral providing  evidence  for  possible  separate  as  well  as  group  recruitment.  Axons  of  canal  efferents 
traveled  anteriorally  for  250  ^m  in  a  paramedian  dorsal  trajectory  before  turning  laterally  to  cross  the 
medullary  tegmentum  and  exit  the  brainstem.  Most  saccular  efferents  pursued  the  same  course  but  about 
10%  followed  a  more  ventral  trajectory  near  the  median  longitudinal  fasiculi  before  they  ascended  to 
join  the  above  bundle.  Somata  of  canal  efferents  were  antidromically  identified  and  were  penetrated  with 
glass  microelectrodes  containing  horseradish  peroxidase.  Most  canal  efferent  neurons  were  only  antidrom- 
ically activated  from  one  peripheral  site  indicating  separate  populations  of  efferents  to  each  canal.  Strad- 
dling of  the  antidromic  stimulus  revealed  underlying  short  latency  depolarizations  that  were  shown  to 
be  indicative  of  electrical  coupling.  Coupling  was  predominantly  limited  to  neurons  from  the  same  canal. 
Efferents  were  spontaneously  active  and  discharged  with  much  higher  frequency  when  the  fish  was  roused 
to  movement  from  any  sensory  stimulus.  The  above  patterns  of  efferent  physiological  activity  suggest 
several  roles  consistent  with  putative  inhibitory  action  on  hair  cells.  Activity  in  the  absence  of  movement 
indicates  a  tonic  modulatory  influence,  and  their  strong  recruitment  associated  with  movement,  another 
type  of  regulatory  mechanism. 

Supported  by  N.I.H.  N.S.  15218. 

Correlation  of  electron  microscopic  fine  structure  with  videomicroscopic  observations 
in  identified  lobster  axons  during  glut  araldehyde  fixation.  A.  J.  HODGE  (Labo- 
ratory of  Biophysics,  NINCDS,  MBL),  C.  K.  GOVIND,  R.  J.  LASEK,  AND  R.  D. 
ALLEN. 


A  preparation  i.  ntaining  two  excitatory  motor  axons  (^30  nm  in  diameter)  from  the  claw  closer 
muscle  of  a  juvenile  ;er  was  observed  by  video-enhanced  AVEC-POL  and  AVEC-DIC  microscopy. 
In  one  of  these  axons,  li  isual  long  mitochondria  were  present  and  moving  normally.  Both  orthograde 
and  retrograde  transport  were  also  clearly  visible  against  a  reasonably  well-resolved  background  of  fila- 
mentous elements  (microtubules)  and  Brownian  movement  was  not  obvious.  The  neighboring  axon, 
however,  contained  immobile  abnormal  appearing  mitochondria  with  blebs,  many  "particles"  showing 
considerable  Brownian  movement,  and  there  were  no  indications  of  transport.  In  all  likelihood,  this  axon 
was  "dead."  Both  axons  were  located  relative  to  an  easily  recognizable  feature  in  the  surrounding  con- 
nective tissue,  and  the  axons  were  then  externally  irrigated  with  an  isotonic  fixative.  The  net  result  of 
this  was  the  cessation  of  all  movement  without  any  detectable  change  in  optical  properties,  and  a  decrease 
in  Brownian  movement. 


NEUROBIOLOGY  385 

The  fixed  preparation  was  subjected  to  routine  post-fixation  with  OsO4,  acetone  dehydration,  and 
embedding  in  Epon  812.  Relatively  thick  (=^0.2  ^m)  transverse  sections  were  observed  in  stereo  using 
a  Philips  EM400  electron  microscope.  Clear-cut  differences  between  the  two  axons  were  seen  in  transverse 
sections.  One  definitely  exhibited  the  "normal  structure"  already  established  for  lobster  and  other  ar- 
thropod axons,  i.e.,  it  contained  a  well-ordered  neuroplasmic  lattice  consisting  of  neurotubules  linked 
transversely  by  periodically  disposed  cross-bridges  and  the  usual  complement  of  organelles  and  small 
vesicles.  The  other  axon  appeared  rather  degraded  by  the  same  criteria.  The  neuroplasmic  lattice  showed 
considerable  deterioration,  and  numerous  vacuoles  were  present  in  the  axoplasm.  The  results  indicate 
that  the  cross-linking  activity  of  glutaraldehyde  does  very  little  other  than  to  maintain  the  structural 
integrity  of  the  axoplasm,  at  least  insofar  as  its  optical  properties  (DIC)  are  concerned.  These  results  are 
in  accord  with  published  x-ray  diffraction  observations  on  protein  crystals  and  paracrystalline  arrays 
(myelin  sheath)  showing  that  the  net  effect  of  glutaraldehyde  fixation  is  the  addition  of  small  bridging 
elements  without  appreciable  loss  of  order. 

Organization  of  mononeuronal  pools  innervating  muscles  of  the  free  fin  rays  in  the 
searobin,  Prionotus  carolinus.  KATHERINE  KALIL  (University  of  Wisconsin)  AND 
THOMAS  E.  FINGER. 

Searobins  (Prionotus  carolinus)  possess  3  pairs  of  fin  rays  used  for  exploratory  movements.  Each 
fin  ray  is  moved  independently  by  a  pair  of  muscles,  an  elevator  and  a  depressor.  The  rostral  spinal  cord 
of  the  searobin  has  3  pairs  of  enlargements  of  the  dorsal  horn  termed  accessory  lobes.  Previous  experiments 
(Finger  1982,  Biol.  Bull.  163:  154-161)  established  that  the  sensory  nerve  to  each  fin  ray  terminates  in 
a  single  accessory  lobe.  These  projections  are  arranged  somatotopically  such  that  the  ventralmost  fin  ray 
is  represented  within  the  caudalmost  lobe,  while  the  pectoral  fin  is  represented  rostral  to  the  lobes.  The 
present  experiments  were  carried  out  to  determine  the  organization  of  the  motoneuronal  pools  innervating 
the  fin  ray  and  pectoral  fin  muscles  and  the  extent  to  which  this  pattern  corresponds  to  that  of  the  sensory 
projections. 

Injections  of  HRP  into  individual  fin  ray  muscles  showed  that  the  motoneuronal  pools  are  discrete 
for  each  fin  ray  and  lie  ventral  to  the  accessory  lobes.  The  motor  pools  are  arranged  in  a  somatotopic 
order  similar  to  the  sensory  projections.  That  is,  the  ventralmost  fin  ray  is  innervated  by  the  caudalmost 
motoneurons  whereas  the  pectoral  fin  motoneurons  lie  rostral  to  the  accessory  lobes.  However,  motor 
neurons  are  not  in  precise  register  with  the  lobes;  rather,  the  motoneuronal  pool  innervating  a  given  fin 
ray  is  shifted  forward  of  the  corresponding  sensory  projection  by  a  distance  approximately  equal  to  one 
half  a  lobe. 

There  are  no  obvious  differences  in  the  location,  numbers,  or  sizes  of  the  motoneurons  innervating 
the  two  different  muscles  of  each  fin  ray.  Moreover,  all  of  the  retrogradely  labeled  neurons  lie  in  the 
ventral  motor  cell  column.  The  unlabeled  dorsal  motoneurons  may  innervate  the  epaxial  or  dorsal  fin 
erector  muscles. 

These  results  coupled  with  previous  studies  indicate  the  possibility  of  a  local  reflex  pathway  for  each 
fin  ray. 

Supported  by  NIH  grant  NS- 14428  (K.K.)  and  NSF  grant  (T.E.F.). 

Light-evoked  field  potentials  and  [K+]0  in  the  skate  retina:  pharmacological  studies 
on  the  cellular  origins  of  the  responses.  C.  J.  KARWOSKI  (University  of  Georgia), 
R.  L.  CHAPPELL,  L.  M.  PROENZA,  R.  B.  SZAMIER,  D.  J.  TAATJES,  V.  MANCINI, 
AND  H.  RIPPS. 

There  is  good  evidence  that  the  most  prominent  electrical  potentials  that  comprise  the  transretinally 
recorded  electroretinogram  (ERG)  result  from  light-induced  changes  in  extracellular  potassium  [K+]0 
acting  passively  on  membranes  of  non-neuronal  elements.  For  example,  the  light-evoked  decrease  in 
[K+]0  recorded  in  the  region  of  photoreceptor  inner  segments  hyperpolarizes  apical  membranes  of  pigment 
epithelial  cells  which,  in  turn,  generate  the  slow  vitreous-positive  c-wave.  A  light-evoked  increase  in  [K+]0 
seen  more  proximally  in  the  retina  is  thought  to  give  rise  to  Miiller-cell  currents  resulting  in  the  earlier, 
more  transient  b-wave.  In  addition,  it  has  been  suggested  that  the  distal  decrease  in  [K+]0  acts  also  on 
the  Miiller  cell  to  produce  a  transretinal  potential  similar  in  time  course  to  the  c-wave  but  opposite  in 
sign,  i.e.,  the  slow  PHI  component  of  the  ERG.  The  results  we  have  obtained  using  various  pharmacologi- 
cal agents  and  recording  with  conventional  and  K+-selective  electrodes  support  the  view  that  slow  PIII 
is  a  K+-dependent  response,  but  they  are  not  consistent  with  the  notion  that  it  originates  across  the 
Miiller-cell  membrane. 

Adding  the  potent  gliotoxin  DL-«-aminoadipic  acid  (a-AAA)  to  perfusate  bathing  the  skate  eyecup 
severely  (and  selectively)  disrupts  the  structural  integrity  of  the  Miiller-cell  membrane  and  disperses  its 
cytoplasmic  contents.  Although  50  mAf  a-AAA  abolished  the  b-wave,  it  did  not  affect  the  distal  decrease 
in  [K+]0,  the  c-wave  or  slow  PIII. 


386  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

The  distal  decrease  in  [K.+]0  was  also  insensitive  to  1.0  mM  Ba2+,  which  eliminated  both  the  c-wave 
and  slow  PHI,  leaving  intact  the  b-wave.  Since  Ba2+  exerted  no  effect  on  this  decrease  of  [K+]0,  these 
findings  indicate  that  changes  in  electrical  activity  due  to  Ba2+  result  from  direct  action  of  this  agent  on 
pigment  epithelial  cells  and  on  the  (unspecified)  cellular  generators  of  slow  PHI.  On  the  other  hand,  the 
fact  that  slow  PHI  and  c-wave  were  suppressed  by  Ba2+,  whereas  the  b-wave  was  unaffected,  suggests  that 
there  is  a  fundamental  difference  in  the  mechanisms  by  which  these  field  potentials  are  generated,  e.g. 
in  the  nature  of  the  K+-channels  of  the  cells  subserving  these  responses. 

This  work  was  supported  by  research  grants  EY-00777,  EY-02988,  EY-03526,  and  EY-00285  from 
the  National  Eye  Institute,  U.  S.  Public  Health  Service,  and  an  award  from  the  Burroughs  Wellcome 
Company  to  Fight  for  Sight,  Inc.,  New  York  City. 

Efferent  neurotransmission  ofcircadian  rhythms  in  Limulus  lateral  eye:  single  cell 
studies.  LEONARD  KASS  AND  ROBERT  B.  BARLOW,  JR.  (Syracuse  University). 

A  circadian  clock  in  Limulus  brain  generates  efferent  lateral  optic  nerve  fibers  at  night.  The  efferent 
fibers  terminate  in  the  retina  and  presumably  release  one  or  more  neurotransmitters  that  mediate  nu- 
merous changes  in  retinal  structure  and  function  (Science  197:  86-89,  1977;  Science  210: 
1037-1039,  1980). 

Long-term  recordings  from  single  optic  nerve  fiber  afferents  indicate  that  at  night  the  steady-state 
response  characteristics  change  in  3  ways:  ( 1 )  spontaneous  spike  activity  is  lowered;  (2)  quantum  catch, 
or  sensitivity,  is  increased;  and  (3)  gain,  or  response  per  photon,  is  increased  (Science  197:  86-89,  1977). 
We  report  that  octopamine  ( 1  nM),  forskolin  ( 10  nM)-  and  dibutyrl-cAMP  ( 100  ^Af)  injected  subcorneally 
into  the  lateral  eye  in  situ  during  the  day  induce  all  3  changes  in  the  optic  nerve  response.  Intracellular 
recordings  from  single  photoreceptor  cells  show  that  at  night  the  frequency  of  spontaneous  fluctuations 
in  membrane  voltage  (dark  bumps)  decrease  whereas  the  response  to  light  increases  (Nature  286:  393- 
395,  1980).  Subcorneal  injection  of  octopamine  during  the  day  reproduces  these  changes. 

Octopamine  has  met  all  five  criteria  for  efferent  neurotransmission  in  Limulus  lateral  eye:  synthesis, 
localization,  and  release  (Science  216:  1250-1252,  1982);  physiological  mimicry  and  pharmacological 
blockade  (Biol.  Bull.  159:  487,  1980;  Biol.  Bull.  161:  348,  1981).  cAMP  may  function  as  a  secondary 
transmitter.  Forskoline,  a  putative  adenylate  cyclase  activator  and  dibutyrl-cAMP  both  change  retinal 
structure  and  physiology  in  a  manner  similar  to  the  octopamine-induced  changes. 

Supported  by  NIH  grants  EY-00667  and  EY-05443  and  NSF  grant  BNS  81-19436. 

Colchicine  blocks  nerve  excitation:  an  optical  study.  DAVID  LANDOWNE  (University 
of  Miami),  JAMES  LARSEN,  AND  KEVIN  TAYLOR. 

Internal  application  of  30  mM  colchicine  to  perfused,  voltage-clamped  squid  axons  produced  a 
rapid,  specific,  and  reversible  decrease  in  sodium  current  to  about  one-third  of  control  values.  The  change 
in  axon  birefringence  which  normally  occurs  when  the  membrane  is  depolarized  was  also  dramatically 
and  reversibly  decreased  by  colchicine.  The  birefringence  response  to  a  hyperpolarizing  pulse  showed 
only  a  slight  decrease.  Application  of  10  mM  colchicine,  had  similar  but  lesser  effects  on  both  the  electrical 
and  the  optical  responses.  A  saturated  (less  than  10  mM)  solution  of  beta-lumicolchicine  also  had  similar 
effects  on  both  the  electrical  and  optical  responses. 

Tetrodotoxin,  applied  externally,  completely  blocked  the  sodium  current  but  did  not  alter  the  bi- 
refringence response  either  in  the  presence  or  absence  of  colchicine. 

The  effect  of  colchicine  is  to  remove  or  slow  an  early  component  of  the  birefringence  response. 
These  experiments  clearly  demonstrate  an  association  of  this  component  with  the  sodium  conductance 
change.  The  site  of  colchicine  action  is  distinct  from  that  of  tetrodotoxin  action  as  seen  from  their 
different  effects  on  the  optical  recordings.  The  direct  involvement  of  microtubules  is  unlikely  in  view  of 
the  lumicolchicine  results. 

Supported  by  NS1 37809.  We  thank  I.  Llano  for  sharing  her  finding  that  colchicine  blocks  the  sodium 
current. 

The  carbon  fiber  electrode:  its  construction  and  use  in  squid  axons.  JAMES  B.  LARSEN 
(University  of  Southern  Mississippi)  AND  DAVID  LANDOWNE. 

Bundles  of  carbon  fibers  are  a  superior  alternative  to  the  platinized  platinum  wire  commonly  used 
in  voltage-clamp  electrodes  Such  fibers  are  uniformly  straight,  easily  manipulated,  and  rebound  without 
damage  after  being  flexed.  Since  complex  surface  preparation  is  not  necessary,  carbon  fiber  electrodes 
can  be  assembled  quickly  with  a  minimum  of  experience.  Following  mild  oxidation  the  current-carrying 
capacity  of  fiber  bundles  is  at  least  equivalent  to  platinized  platinum  of  equal  diameter. 


NEUROBIOLOGY  387 

In  our  electrodes,  current  is  carried  by  a  bundle  of  1 8-20  carbon  fibers,  each  having  a  diameter  of 
10  ^m  (Thornel  P-55;  Union  Carbide  Corp.).  This  is  attached  with  epoxy  to  the  voltage  electrode,  which 
is  a  microcapillary  of  fused  silica  containing  a  fine  platinum  wire,  mounted  in  the  tip  of  a  platinum 
syringe  needle  insulated  with  a  polyethylene  sleeve.  The  needle  imparts  structural  strength  to  the  entire 
electrode  assembly  and  improves  the  response  of  the  voltage  electrode.  Electrical  contact  between  a  copper 
wire  and  the  carbon  fibers  is  made  with  conductive  paint  containing  silver.  Spurious  current  flow  from 
exposed  silver  and  platinum  surfaces  is  prevented  with  a  coating  of  epoxy.  After  assembly,  electrodes  are 
oxidized  in  25  mM  citric  acid,  titrated  to  pH  5.2  with  NaOH,  for  5  min. 

Our  experience  reveals  no  change  in  electrode  performance  during  two  months  of  daily  use.  Analysis 
of  current  traces  from  typical  voltage-clamp  experiments  suggests  that  carbon  fiber  electrodes  will  support 
current  densities  of  at  least  0.26  mA/cm2  in  a  400  ^m  squid  axon,  for  each  fiber  included  in  the  bundle. 

This  work  was  supported  by  NIH  grant  NS 137809.  We  thank  A.  Strickholm  for  his  kind  gift  of 
carbon  fibers. 

Synthesis  and  release  of 3 H-octopamine  from  the  cardiac  ganglion  of  Limulus  po- 
lyphemus.  S.  C.  LUMMIS  (Cambridge  Univ.,  UK),  P.  M.  O'CONNOR,  AND 
B.  A.  BATTELLE. 

Octopamine,  a  biogenic  phenolamine,  is  a  likely  candidate  as  a  neurotransmitter  or  neurohormone 
in  the  cardiac  ganglion  of  Limulus  polyphemus  (Augustine  et  al.  1982,  J.  Neurobiol.  13:  61-74).  We 
report  that  the  cardiac  ganglion  can  synthesize  octopamine  from  3H-tyramine  and  that  this  newly  syn- 
thesized octopamine  can  be  released  by  depolarizing  agents.  Octopamine  in  the  cardiac  ganglion  was 
identified  using  high  voltage  paper  electrophoresis,  and  the  release  of  3H-metabolites  from  isolated  gan- 
glion was  monitored  by  liquid  scintillation  counting.  The  cardiac  ganglion  synthesized  an  average 
(n  =  3)  of  10.4  picomoles  octopamine/mg  wet  weight  tissue  when  incubated  in  medium  containing  10 
nd  3H-tyramine/ml.  In  addition  to  3H-octopamine,  two  unidentified  radiolabeled  metabolites  were  de- 
tected in  the  acid  extract  of  the  ganglion.  The  cardiac  ganglion  released  3H-octopamine  when  stimulated 
by  either  200  mM  K.C1  or  50  nM  veratridine.  The  veratridine-induced  release  was  prolonged  relative  to 
the  KCl-induced  release  and  exhibited  a  delayed  onset  of  maximum  response.  In  addition,  veratridine 
induced  a  release  of  one  of  the  unidentified  metabolites.  The  effects  of  both  KC1  and  veratridine  were 
blocked  by  preincubating  the  ganglion  with  40  mM  CoCl2,  suggesting  a  Ca++-dependent  release  mech- 
anism. The  veratridine-induced  release  was  also  demonstrated  to  be  Na+  dependent:  release  was  blocked 
by  Na+-free  saline.  In  summary,  our  results  are  consistent  with  the  hypothesis  of  a  neuroregulatory  role 
for  octopamine  in  the  cardiac  ganglion  of  Limulus. 

Supported  in  part  by  NIH  Training  Grant  T32  NS  07 165  and  a  Grass  Foundation  grant  to  the  MBL 
summer  Neurobiology  Course. 

Paracrystalline  arrays  of  neurofi lament  protein.  JANIS  METUZALS,  DAVID  F.  CLAPIN 
(Faculty  of  Heaith  Sciences,  University  of  Ottawa,  Ottawa  KIH  8M5,  Ontario, 
Canada),  GLENN  J.  FENNELLY,  AND  PETER  A.  M.  EAGLES. 

Studies  of  paracrystalline  arrays  of  cytoskeletal  proteins  have  contributed  substantially  to  the  knowl- 
edge of  the  properties  of  these  proteins  and  their  interactions.  We  are  reporting  results  of  experiments 
on  formation  of  characteristic  paracrystalline  arrays  of  neurofilament  protein  isolated  from  axoplasm  of 
squid  giant  nerve  fiber.  The  extruded  axoplasm  rods  were  extracted  for  up  to  twelve  hours  at  room 
temperature  in  the  following  solution:  300  mM  potassium  methanesulfonate,  150  mA/taurine,  100  mM 
potassium  glutamate,  12.9  mM  MgCl2,  5  mM  ATP,  3  mMCaCl2,  10  mM  EGTA,  10  mM  MOPS,  pH 
7.2.  Analysis  of  the  rods  by  SDS-PAGE  demonstrated  the  presence  of  two  major  peptides  (200  and  60K) 
and  a  minor  band  at  223K.  SEM  and  TEM  confirmed  the  presence  of  10  nm  neurofilaments  and  their 
finer,  cross-linking  structures. 

A  series  of  recrystallization  experiments  was  patterned  after  the  procedure  used  for  the  preparation 
of  tropomyosin  paracrystals.  Neurofilament  rods  (10  to  18)  were  homogenized  in  300  n\  of  0.5  M  KC1, 
0.5  M  Na2HPO4,  pH  7.0  in  a  chilled  glass-teflon  homogenizer.  The  homogenate  was  dialyzed  overnight 
against  1  liter  of  0.05  M  Tris,  pH  8.0  at  4°C.  The  dialysis  was  continued  against  2  liters  of  0.12  M 
(NH4)2SO4,  0.01  M  sodium  acetate,  pH  5.4  for  10  h  at  4°C.  Electron  microscopy  of  negatively  stained 
retentate  revealed  a  continuous  network  of  collapsed  tubes  (diameter  300  nm-1  nm)  and  rope-like  strands 
(diameter  10  nm-50  nm).  The  tubes  consisted  of  2-nm  wide  unit-filaments  intercoiled  to  comprise  10- 
nm  neurofilaments  which  are  cross-associated  into  a  network.  The  neurofilaments  are  oriented  at  narrow 
angles  against  the  transverse  axis  of  the  tube  producing  patterns  of  overlapping  striations.  Such  patterns 
may  result  from  superposition  of  helically  ordered  filaments  of  the  upper  and  lower  wall  of  the  collapsed 


388  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

tube.  There  is  a  dark  line  coinciding  with  the  central  axis  of  many  tubes.  Analysis  of  the  diffraction 
pattern  of  the  electron  micrographs  of  the  tubes  indicated  periodicities  ranging  from  20  to  60  nm. 
Numerous,  randomized,  individual  10-nm  filaments  were  also  observed. 

The  observed  paracrystalline  arrays  are  expressions  of  intrinsic  properties  of  the  neurofilament 
protein:  phase  transition,  association  in  networks  and  helicity. 

This  investigation  was  supported  by  grant  MA- 1247  from  the  Medical  Research  Council  of  Canada. 

Quantitative  aspects  of  growth  of  an  identified  neuron  in  the  leech  Hirudo  medicinalis. 
MICHELE  MUSACCHIO  AND  EDUARDO  R.  MACAGNO  (Columbia  University). 

We  have  studied  quantitatively  post-embryonic  growth  of  an  identified  neuron  in  the  leech  using 
dye-injection  and  techniques  of  computer  reconstruction.  Each  segmental  ganglion  has  three  pairs  of 
touch  sensory  neurons  (T  cells),  one  with  receptive  fields  on  dorsal  skin  of  the  corresponding  segment. 
Six  dorsal  T  cells  from  two  adult  (three-  to  four-year-old)  animals  and  six  from  a  juvenile  one-year-old 
were  studied.  The  cells  were  in  ganglia  7  through  10.  The  lengths  of  secondary  branches,  their  number 
and  distribution  along  the  main  axon,  the  number  of  synaptic  varicosities,  and  the  total  volume  of 
neuropil  innervated  by  the  cell  were  measured.  The  number  of  branches  varied  from  cell  to  cell,  as  did 
their  average  lengths  within  each  cell,  by  greater  than  20%.  However,  the  total  length  of  secondary 
branching  was  relatively  constant  (within  15%)  among  juvenile  and  among  adult  cells.  The  values  of  all 
the  parameters  measured  increased  from  the  juvenile  to  the  adults.  The  average  number  of  branches 
increased  by  45%,  their  average  length  by  50%,  and  the  total  length  of  branching  approximately  doubled. 
The  number  of  varicosities  increased  by  58%,  and  the  volume  of  neuropil  innervated  tripled.  These 
changes  result  in  an  adult  T  cell  with  a  lower  density  of  innervation  of  the  neuropil  than  the 
juvenile  cell. 

The  data  raise  several  questions  about  the  significance  of  structural  changes  during  the  growth  of 
the  T  cell.  The  additional  varicosities  may  represent  a  strengthening  of  existing  connections  or  the 
establishment  of  new  contacts  with  other  post-synaptic  targets.  New  branches  may  be  inserted  all  along 
the  main  processes  or  only  in  special  regions.  Reduced  density  of  innervation  by  the  T  cell  could  reflect 
increased  innervation  by  other  neurons  or  increased  occupation  of  neuropil  by  glial  processes. 

Intracellular  staining  with  potentiometric  dyes:  optical  signals  from  identified  leech 
neurons  and  their  processes.  A.  L.  OBAID,  H.  SHIMIZU,  AND  B.  M.  SALZBERG 
(University  of  Pennsylvania). 

Using  a  fluorescent  potentiometric  dye,  injected  iontophoretically,  we  have  been  able  to  record 
selectively  and  without  signal  averaging,  optical  signals  corresponding  to  action  potentials  in  somata  and 
main  processes  of  identified  sensory  neurons  of  the  leech,  Hirudo  medicinalis.  Lateral  P  and  N  cells  were 
filled  with  a  positively  charged  styryl  dye,  RH  461  (see  Grinvald  el  al.  Biol.  Bull.  163:  383)  by  means 
of  250  msec,  0.5  nA  current  pulses  (50%  duty  cycle)  applied  for  five  minutes.  Following  injection, 
illumination  of  the  ganglion  with  the  green  portion  (interference  filter  540  ±  15  nm)  of  the  output  of 
an  electronically  shuttered  high  pressure  mercury  arc  (Osram  HBO  100W/2)  revealed  a  strong  red  flu- 
orescence from  the  soma  and  processes.  A  suction  electrode  on  the  ipsilateral  posterior  root  was  used 
to  stimulate  the  injected  cell  whose  main  process  is  directed  out  the  ipsilateral  anterior  root. 

The  ganglion  was  pinned  in  a  chamber  mounted  on  the  stage  of  a  modified  Reichert  Zetopan 
microscope  having  focusable  stage  and  head.  Epi-illumination  with  a  40X,  0.75  n.a.  water  immersion 
objective  (Zeiss)  produced  a  real  image  above  the  trinocular  tube.  A  single  photodiode  (E.G.&G. 
PV-444)  could  be  positioned  behind  a  set  of  four  independent  knife  edges,  permitting  the  optical  isolation 
of  a  region  of  the  preparation  of  arbitrary  size,  aspect  ratio,  and  orientation.  The  fluorescence  emission 
from  the  injected  cell  was  selected  by  means  of  a  Zeiss  dichroic  mirror  (FT580)  and  a  Schott  glass  barrier 
filter  (RG630).  Excess  noise  from  the  arc  was  reduced  by  means  of  a  reference  photodiode,  located 
beneath  the  preparation,  which  sampled  the  transmitted  intensity.  The  gain  of  its  photocurrent-to- voltage 
converter  was  continuously  variable,  and  its  DC  output  could  be  matched,  by  transient  nulling,  with 
that  of  the  fluorescence  detector.  The  AC  coupled  outputs  of  the  two  photodetectors  were  then  measured 
differentially.  A  response  time  constant  of  600  ^sec  and  AC  coupling  time  constant  of  100  msec  were 
employed. 

This  technique  may  be  used  to  study  the  cable  properties  of  extrasomatic  regions  of  cells,  and  to 
monitor  the  invasion  of  their  branches  and  large  terminals  and  the  integration  of  information  in  neuronal 
arborizations. 

We  are  most  grateful  to  A.  Grinvald  and  R.  Hildesheim  for  the  synthesis  and  gift  of  RH  461. 


NEUROBIOLOGY  389 

Supported  by  U.S.P.H.S.  grant  NS  16824,  A  STEPS  Fellowship  to  A.L.O.,  and  a  grant  from  the  Agency 
of  Science  and  Technology  (Japan)  to  H.S. 

/ 

/ 

Optical  monitoring  of  evoked  activity  in  the  visual  cortex  of  the  marine  rat.  H.  S. 
ORBACH  (Dept.  of  Physiology,  Yale  University),  L.  B.  COHEN,  AND  A.  GRIN- 
VALD. 

Recent  experiments  on  the  salamander  olfactory  bulb  and  goldfish  optic  tectum  showed  that  optical 
methods  could  be  used  to  monitor  electrical  activity  in  these  preparations.  We  wanted  to  determine  if 
the  same  method  would  be  applicable  to  the  mammalian  cortex. 

We  began  by  staining  tests  using  1 6  fluorescent  dyes  that  were  known  to  give  relatively  large  potential- 
dependent  signals  in  squid  axons  to  see  if  they  would  stain  and  penetrate  into  the  rat  brain  when  applied 
in  concentrated  solutions  (0.1-1  mg/ml)  to  the  surface  of  the  cortex.  Two  pyrazolone-oxonol  dyes  pen- 
etrated 2  mm  and  four  styryl  dyes  penetrated  200-300  ^m  after  a  one  or  two  hour  staining  period.  These 
dyes  were  then  tested  in  optical  experiments  where  visual  cortex  was  exposed  and  stained;  simultaneous 
measurements  of  fluorescence  were  made  from  124  cortical  loci  using  epi-illumination  and  a  124  element 
diode  array  on  a  Leitz  Ortholux  II  microscope.  Two  kinds  of  stimulation  were  used.  Either  a  20  msec 
light  flash  was  delivered  to  the  intact  eye  or  the  eye  was  removed  and  a  suction  electrode  was  used  to 
stimulate  the  optic  nerve  directly. 

Optical  signals  from  the  cortex  were  found  in  response  to  both  kinds  of  stimulation.  The  signals  in 
response  to  optic  nerve  stimulation  were  relatively  large,  AF/F  was  1-3  X  10~3,  and  reached  a  peak  20 
msec  after  the  stimulus.  The  signals  in  response  to  light  stimulation  reached  a  peak  about  60  msec  after 
the  beginning  of  the  light  flash.  Measurements  at  wavelengths  outside  the  absorption  band  of  the  dyes 
did  not  give  rise  to  signals;  thus  the  signals  were  not  the  result  of  light  scattering  changes  or  mechanical 
artifacts. 

Although  additional  experiments  are  needed  to  determine  the  localization  of  these  signals  to  specific 
areas  of  cortex,  our  results  suggest  that  optical  methods  may  provide  a  powerful  tool  for  monitoring 
activity  in  many  cortical  sites  simultaneously.  We  think  that  such  a  method  could  be  useful  in  studying 
cortical  organization. 

Supported  by  N.I.H.  grants  NS08437  and  NS  14716  and  a  grant  from  the  U.S.-Israel  Binational 
Science  Foundation. 


Asymmetry  in  the  olfactory  system  of  the  winter  flounder,  Pseudopleuronectes  amer- 
icanus.  P.  D.  PRASADA  RAO,  THOMAS  E.  FINGER  (Univ.  Colorado  Medical 
School),  AND  WAYNE  L.  SILVER. 

During  metamorphosis  in  the  winter  flounder,  the  left  eye  migrates  to  the  right  side  of  the  head  so 
that  both  eyes  come  to  lie  on  the  upper  side  of  the  fish.  Although  migrated,  the  eyes  maintain  symmetry 
in  their  projections  into  the  central  nervous  system  (Luckenbill-Edds  and  Sharma  1977,  J.  Comp.  Neural. 
173:  307-318).  However,  the  olfactory  organs  do  not  migrate  far  from  their  original  position,  so  in  the 
adult  the  right  olfactory  organ  is  located  on  the  upper  side  while  the  left  is  turned  partially  towards  the 
substratum.  Concomitantly,  the  upward-facing  organ  comprises  1 1  or  12  large  lamellae  whereas  the  other 
organ  consists  of  only  5  or  6  smaller  lamellae.  Also,  the  right  olfactory  nerve  is  thicker,  and  the  right 
olfactory  bulb  approximately  three  times  larger,  than  their  contralateral  counterparts. 

Horseradish  peroxidase  was  used  as  a  neuronal  tracer  to  compare  the  central  projections  of  the  right 
and  left  olfactory  bulbs.  The  overall  pattern  of  olfactory  bulb  projections  into  the  prosencephalon  in  the 
flounder  did  not  differ  markedly  from  that  reported  in  other  teleost  species.  However,  the  fiber  bundle 
extending  from  the  larger  olfactory  bulb  into  the  contralateral  telencephalon  via  the  anterior  commissure 
is  thicker  than  its  counterpart  arising  from  the  small  olfactory  bulb.  The  terminal  fields  of  the  large  (right) 
olfactory  bulb  in  the  dorsal,  ventral,  and  posterior  areas  of  both  the  ipsilateral  and  contralateral  telen- 
cephalon are  quite  extensive.  In  contrast,  the  projections  of  the  left  olfactory  bulb  into  the  ipsilateral 
telencephalon  are  less  elaborate,  and  the  contralateral  terminal  fields  are  relatively  sparse. 

The  more  extensive  projections  of  the  right  olfactory  bulb  are  associated  with  the  greater  development 
of  the  right  olfactory  organ.  The  asymmetric  projections  of  the  olfactory  bulbs  in  the  adult  may  be  due 
to  postmetamorphic  differential  growth  of  the  olfactory  epithelium  and  bulbs  on  the  two  sides  of  the 
flounder. 

Supported  by  NIH  and  NSF  grants  (T.E.F.)  and  a  Grass  Foundation  Fellowship  (W.L.S.). 


390  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Does  the  Schwann  cell  of  Loligo  act  as  a  potassium  electrode?  Optical  studies 
using  potentiometric  probes.  B.  M.  SALZBERG,  A.  L.  OBAID,  H.  SHIMIZU, 
R.  K.  ORKAND,  AND  D.  M.  SENSEMAN  (University  of  Pennsylvania). 

In  common  with  that  of  glial  elements  in  mammalian  systems,  the  physiology  of  the  Schwann  cells 
that  intimately  surround  the  giant  axons  of  squid  is  poorly  understood.  In  Loligo,  electrophysiological 
studies  have  been  limited  to  measurements  of  the  impedance  characteristics  of  the  Schwann  layer — the 
small  size  and  tortuous  geometry  of  the  cells  precluding  transmembrane  voltage  measurements.  In  Se- 
pioteuthis  the  cells  are  3-5  times  thicker,  and  one  laboratory  has  described  a  long-lasting  //y/^/polarization 
of  the  Schwann  cell  in  response  to  rapid  trains  of  impulses.  They  attribute  this  to  nicotinic  cholinergic 
transmission  from  axon  to  Schwann  cell  (Villegas  1974,  J.  Physiol.  242:  647-659).  We  have  attempted 
to  measure  electrical  events  in  the  Schwann  cell  by  exploiting  the  linear  potentiometric  changes  in  light 
absorption  exhibited  by  membrane  stained  with  an  impermeant  merocyanine-oxazolone  dye,  NK  2367. 
Superfused,  the  probe  should  bind  to  Schwann  cell  membrane  and  axolemma,  optical  signals  reflecting 
voltage  changes  in  both  cell  membranes.  Differences  between  the  optical  measurement  and  an  electrode 
recording  should  result  principally  from  potential  variation  in  the  Schwann  cell.  Perfused  internally,  the 
dye  should  reach  only  axolemma,  and  the  optical  signal,  inverted,  should  closely  resemble  an  electrode 
recording.  We  employed  volleys  of  impulses  (250-333  Hz)  to  raise  the  potassium  concentration  in  the 
periaxonal  space  by  about  20  mM,  and  we  simultaneously  monitored  the  Frankenhaeuser-Hodgkin  effect 
optically  and  electrically  (T  =  3.5  jisec).  When  the  probe  was  applied  intracellularly,  the  two  signals  were 
superimposable,  after  scaling.  Extracellular  staining  revealed  a  striking  and  consistent  difference  in  the 
envelopes  of  the  spike  undershoots.  The  optical  record  was  altered  in  the  direction  expected  if  the  Schwann 
cell  underwent  a  very  small  hvperpolarization,  assuming  that  dye  bound  to  glial  membrane  behaves 
optically  as  it  does  when  bound  to  axolemma.  The  effect,  corrected  simply  for  membrane  area,  was  about 
0.7  mV  and  was  not  sensitive  to  curare  (10~6  A/).  Neither  ouabain  nor  strophanthidin  (10  4  M)  reliably 
altered  the  result,  suggesting  that  an  electrogenic  pump  is  not  implicated.  Similar  experiments  on  un- 
stained preparations  disclosed  a  very  small  effect,  resembling  the  difference  signal,  which  depended  upon 
the  time  integral  of  the  outward  current  (Cohen  el  al.  1972,  J.  Physiol.  224:  727-752).  While  much  too 
small  to  explain  our  observations,  this  signal  may  be  enhanced  by  the  extracellular  presence  of  the  dye, 
in  a  manner  dependent  upon  the  geometry  of  the  Schwann  layer  and,  in  particular,  the  Frankenhaeuser- 
Hodgkin  space. 

Supported  by  U.S.P.H.S.  grants  NS  16824,  NS  12253,  DE  05536,  a  STEPS  Fellowship  to  A.L.O. 
and  a  grant  from  the  Agency  of  Science  and  Technology  (Japan)  to  H.S. 

Responses  from  spinally  innervated  chemoreceptors  on  the  free  fin  rays  of  the  sea- 
robin,  Prionotus  carolinus.  WAYNE  L.  SILVER  (Monell  Chemical  Senses  Center) 
AND  THOMAS  E.  FINGER. 

The  spinally  innervated  free  fin  rays  of  the  searobin,  Prionotus,  are  sensitive  to  chemical  stimuli 
despite  the  absence  of  taste  buds  or  olfactory  receptors.  The  present  research  examines  the  sensitivity  of 
the  fin  ray  chemoreceptors  to  a  variety  of  compounds.  Using  conventional  electrophysiological  techniques, 
we  recorded  neural  responses  from  the  fin  ray  nerves  in  immobilized,  artificially  respired  searobins. 
Amino  acids,  because  of  their  abundance  in  searobins'  diet  and  their  extreme  effectiveness  as  olfactory 
and  gustatory  stimuli  in  other  fish,  were  the  principal  compounds  tested. 

Responses  to  mechanical  and  proprioceptive  stimuli  were  observed  in  all  36  searobins  tested,  and 
the  fin  rays  are  apparently  extremely  sensitive  to  touch  and  position.  Responses  to  chemical  stimuli  were 
obtained  in  2 1  of  36  fish.  Chemical  stimuli  elicited  rapidly  adapting  responses.  Betaine  HC1  (trimethyl 
glycine)  was  the  most  effective  compound  tested.  The  order  of  effectiveness  of  other  stimulatory  com- 
pounds at  10  2  A/ was:  dimethyl  glycine  HC1  (DMG)  >  L-a-ABA  >  gly  >  L-ala  >  L-cysh  >  L-pro  >  L- 
thr  >  L-ser  >  L-arg  >  L-phe.  In  addition,  squid  extract  (1  g/ 100  ml  artificial  sea  water)  was  an  extremely 
effective  stimulus.  Of  the  18  amino  acids  in  squid  extract,  betaine,  gly,  L-ala,  and  L-pro  are  among  the 
five  found  in  highest  concentration  (Mackie,  1982  in  Chemoreception  in  Fishes,  T.  J.  Hara,  ed.  pp.  275- 
291).  Compounds  tested  which  did  not  elicit  a  response  at  10  2  M  included  L-gln,  L-glu,  sucrose  (1.0 
M),  taurine,  trimethylamine  oxide,  choline  chloride,  and  acetic  acid  (0.02  M).  The  lowest  threshold  was 
to  betaine(10-55  M),  followed  by  DMG(10-40  A/),  L-a-ABA(10-40 M),  L-ala(10~3  :5  M),  gly(  1 0'3 °  A/), 
L-thr(10^30Af),  L-ser(10~20A/),  and  L-phe(10-20A/). 

The  results  show  that  chemoreceptors  on  the  free  fin  rays  of  searobins  respond  to  relatively  low 
concentrations  of  certain  amino  acids.  The  variety  and  threshold  concentrations  of  stimulatory  amino 
acids  resemble  those  reported  for  the  taste  systems  of  other  marine  teleosts  (Kiyohara  et  al.  1975,  Bull. 
Jpn.  Soc.  Sci.  Fish.  41:  383-391).  These  results  indicate  that  the  chemoreceptors  on  the  free  fin  rays  of 
searobins  are  responsive  to  compounds  particularly  prevalent  in  the  animal's  natural  diet. 

Supported  by  a  Grass  Foundation  Fellowship  (W.L.S.)  and  an  NSF  grant  (T.E.F.). 


PARASITOLOGY  AND  PATHOLOGY  39 1 

PARASITOLOGY  AND  PATHOLOGY 

Stage-specific  gene  expression  in  Plasmodium  gallinaceum.  JAY  BANGS, 
STEVEN  ZEICHNER,  ROBERT  BARKER,  RICHARD  CARTER,  AND  DYANN  WIRTH 
(Harvard  School  of  Public  Health). 

P.  gallinaceum,  an  avian  malaria,  undergoes  transformation  during  sexual  differentiation  that  in- 
volves extensive  morphological  changes  in  a  brief  time  period.  In  these  experiments  we  examined  the 
biosynthesis  and  processing  of  sexual  stage-specific  proteins.  The  gametocytes  of  P.  gallinaceum  can  be 
induced  to  exflagellate  and  differentiate  through  the  sexual  cycle  as  far  as  the  ookinete  stage  in  vitro. 
Blood  was  obtained  from  infected  chickens,  washed,  and  pulse-labeled  in  vitro  with  35S-methionine  under 
conditions  preventing  exflagellation.  Labeled  parasites  were  washed  and  divided  into  two  groups.  One 
group  was  allowed  to  exflagellate,  the  other  was  not.  Samples  were  taken  immediately  after  the  pulse, 
at  1  h  and  at  5  h  of  chase,  and  lysed  in  1%  Triton  X-100.  Aliquots  of  each  lysate  were  analysed  for  total 
labeled  protein  by  SDS-PAGE.  The  remainder  of  each  lysate  was  divided  three  ways  and  immunopre- 
cipitated  with  either  normal  rabbit  serum,  rabbit  anti-zygote  serum,  or  rabbit  anti-ookinete  serum.  The 
immunoprecipitates  were  analysed  on  SDS-PAGE  and  the  proteins  detected  by  coomassie  blue  staining 
and  fluorography.  Band  patterns  in  fluorographs  of  both  exflagellated  and  nonexflagellated  pulse  chases 
showed  remarkable  similarity.  Most  bands  showed  no  change  in  intensity  during  the  period  of  incubation. 
However,  one  band  of  Mr  55K.  decreased  in  intensity  while  one  band  of  Mr  36K  increased  in  intensity 
in  both  groups.  These  findings  suggest  possible  protein  processing  during  the  chase  period.  Both  anti- 
zygote  and  anti-ookinete  sera  failed  to  reveal  any  significant  differences  in  protein  labeling  of  exflagellated 
and  non-exflagellated  parasites  at  both  the  1  h  and  5  h  chase  points.  At  1  h  anti-zygote  serum  recognized 
more  proteins  than  anti-ookinete  serum.  At  5  h  this  difference  was  less  evident.  Analysis  of  the  immu- 
noprecipitates revealed  that  zygotes  and  ookinetes  have  several  common  antigens,  in  addition  to  ookinete- 
and  zygote-specific  antigens.  These  data  indicate  that  proteins  recognized  by  antisera  to  zygotes  and 
ookinetes  are  synthesized  and  present,  prior  to  exflagellation,  in  the  erythrocytic  stages. 

Surface  labeling  of  Trypanosoma  cruzi.  TECIA  MARIA  ULISSES  DE  CARVALHO  AND 
MIERCIO  PEREIRA  (Institute  de  Biofisica,  Bloco  G,  Centre  de  Ciencias  da  Saude, 
UFRJ,  Rio  de  Janeiro,  Brasil). 

Epimastigotes  and  trypomastigotes,  two  developmental  forms  of  Trypanosoma  cruzi,  were  surface- 
labeled  with  I25I  using  lodogen  (which  catalyzes  iodination  of  tyrosine  residues)  and  iodonapthylazide 
(INA)  which  labels  membrane  proteins  embedded  in  lipid  bilayer.  Epimastigotes  were  obtained  from 
liquid  cultures  and  trypomastigotes  from  fibroblasts  infected  with  T.  cruzi. 

A  band  of  molecular  weight  75,000  was  present  only  in  epimastigotes,  and  another  of  molecular 
weight  90,000  was  detected  exclusively  in  trypomastigotes  using  the  lodogen  technique,  whereas  bands 
of  molecular  weight  45,000  and  35,000  were  present  in  both  developmental  forms  of  the  trypanosomes. 

Using  iodonapthylazide  reagent,  three  bands  were  identified,  which  showed  identical  molecular 
weights  in  both  forms  of  T.  cruzi.  One  of  them  had  a  molecular  weight  of  35,000  and  this  may  be  identical 
with  the  35,000  mol  wt  band  labeled  by  the  lodogen.  The  other  bands  were  unique  and  had  mol  wt  of 
50,000  and  20,000. 

In  conclusion,  the  results  indicate  that  the  developmental  stages  of  T.  cruzi  have  unique  surface 
proteins  as  detected  by  labeling  tyrosine  residues  and  conserved  proteins  as  determined  by  labeling  the 
integral  membrane  proteins. 

Control  of  tubulin  gene  expression  during  transformation  of  Leishmania  parasites 
from  amastigote  to  promastigote  stages.  MARIE-FRANCE  DELAUW,  SCOTT 
LANDFEAR,  DIANNE  MCMAHON  PRATT,  AND  DYANN  WIRTH  (Dept.  of  Tropical 
Public  Health,  Harvard  School  of  Public  Health,  Boston,  MA). 

Leishmania  parasites  grow  inside  the  macrophages  of  their  mammalian  hosts  as  non-motile  intra- 
cellular  forms  called  amastigotes.  When  infected  macrophages  are  ingested  by  the  sandfly  vector,  the 
parasites  are  released  from  the  macrophages  and  undergo  a  striking  morphological  transformation  to  a 
motile  flagellated  extracellular  form  called  the  promastigote.  K.  P.  Chang  and  colleagues  at  the  Rockefeller 
University  have  shown  that  tubulin  biosynthesis  increases  dramatically  when  amastigotes  are  transformed 
to  promastigotes  in  vitro. 

We  are  studying  the  control  of  expression  of  the  tubulin  genes  in  amastigotes  and  promastigotes. 
We  have  used  a  genomic  clone  of  the  alpha-tubulin  gene  from  Leishmania  enriettii,  isolated  in  the 
laboratory  of  Dr.  Dyann  Wirth,  to  probe  Southern  blots  of  genomic  DNA  from  amastigotes  and  pro- 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

mastigotes.  The  preliminary  results  suggest  that  the  tubulin  genes  may  increase  in  copy  number  upon 
transformation  of  amastigotes  to  promastigotes.  We  are  also  using  a  sea  urchin  beta-tubulin  clone,  ob- 
tained from  Dr.  Joan  Ruderman,  to  determine  whether  the  beta-tubulin  genes  ofLeishmania  are  amplified 
or  rearranged  upon  transformation  from  amastigotes  to  promastigotes. 


A  gill  disease  of  Limulus  polyphemus  associated  with  triclad  turbellarid  worm  in- 
fection. JOSEPH  M.  GROFF  (Laboratory  for  Marine  Animal  Health,  Marine  Bio- 
logical Laboratory)  AND  Louis  LEIBOVITZ. 

Severe  gill  erosions  were  found  to  occur  on  the  book  gills  of  wild  and  captive  Limulus  polyphemus. 
The  pathogenesis  of  these  lesions  and  their  relationship  to  the  triclad  turbellarid  worms,  Bdelloura  Candida 
and  Syncoelidium  pellucidum,  and  their  cocoons  (egg  capsules)  were  investigated. 

Ante-  and  post-mortem  examinations  were  conducted  on  three  groups  of  L.  polyphemus:  six  freshly 
caught  specimens,  six  specimens  held  in  captivity  for  six  weeks,  and  six  morbid  or  dead  captive  specimens 
maintained  in  a  separate  collection  for  an  unknown  period  of  time.  Hematological  studies  were  conducted, 
and  blood  and  gills  were  cultured  for  bacteria.  Tissues  were  taken  for  histopathological  study. 

Normal  gill  lamellar  structure  consisted  of  a  double-walled  three-layered  chitinous  cuticle,  two 
hypodermal  cell  layers,  trabecular  cells,  and  previously  undescribed  multicellular  lamellar  pillar  corpuscles 
with  their  associated  pore-like  structures;  the  latter  two  delineated  the  vascular  channels  within  the  gill 
lamellae.  Cocoons  produced  a  pressure  atrophy  of  the  associated  and  adjacent  gill  lamellae  resulting  in 
degeneration  and  destruction  of  the  gill  cuticle.  Subsequent  changes  within  the  lamellae  included  blood 
loss,  degranulation  of  amebocytes,  blood  coagulation  and  occlusion  of  gill  vascular  channels.  Progressive 
extension  of  these  lesions  resulted  in  gross  gill  erosions  and  perforations.  These  lesions  provided  a  portal 
of  entry  for  immature  worms  and  bacteria. 

This  study  documents  that  cocoons  and  immature,  invasive  triclad  turbellarids  are  pathogens  of  L. 
polyphemus.  Prevention,  control  and  eradication  of  the  disease  in  captive  laboratory  populations  of  L. 
polyphemus  will  be  evaluated  in  future  studies. 

We  wish  to  thank  Ms.  Amy  Stone  for  her  technical  assistance.  This  study  has  been  supported  in 
part  by  a  grant  (No.  1-40-PRO 1333-01)  from  the  Division  of  Research  Resources,  National  Institutes  of 
Health. 

Identification  of  protective  antigens  o/'Schistosoma  mansoni  by  Eastern  blots  using 
monoclonal  antibodies.  R.  PAUL  JOHNSON  AND  DON  HARN  (Harvard  Medical 
School). 

We  have  recently  produced  monoclonal  antibodies  against  the  helminth  Schistosoma  mansoni  (D. 
Harn  et  ai,  in  preparation).  These  monoclonals  bind  to  the  surface  of  schistosomula  as  determined  by 
immunofluorescence  and  produce  a  decrease  in  worm  burden  (40-70%  as  compared  with  controls)  when 
administered  to  mice  prior  to  challenge  with  cercariae  or  schistosomula.  Immunoprecipitation  and  iso- 
electric  focusing  blots  (developed  by  D.H.)  were  used  to  characterize  the  antigens  recognized  by  these 
monoclonals.  Soluble  egg  extracts  and  0.5%  Triton  extracts  of  schistosomula  were  resolved  in  agarose 
isoelectric  focusing  gels,  transferred  to  nitrocellulose  paper  by  wicking,  quenched,  incubated  with  mono- 
clonal culture  supernatants,  chronically  infected  or  non-infected  mouse  sera,  and  probed  with  125I-rabbit 
anti-mouse  immunoglobulin.  Immunoprecipitations  were  performed  with  0.5%  Triton  extracts  (with 
protease  inhibitors)  of  schistosomula  metabolically  labeled  with  35S-methionine  and  Protein  A-Sepharose 
beads  precoated  with  rabbit  anti-mouse  immunoglobulin.  Two  monoclonal  antibodies  recognize  an  egg 
antigen  with  a  pi  of  4.5  and  a  schistosomula  antigen  with  a  pi  of  5.7,  both  of  which  are  also  detected 
by  chronic  infection  sera.  Immunoprecipitation  with  chronic  infection  sera  reveals  antigens  with  molec- 
ular weights  of  225,000,  1 15,000,  85,000,  53,000,  40,000,  and  35,000;  hybridomas  specifically  precipitate 
an  antigen  of  40,000  molecular  weight. 

These  results  demonstrate  that  isoelectric  focusing  blots  (Eastern  blots)  may  be  used  to  characterize 
antigens  recognized  by  monoclonal  antibodies.  The  Eastern  blot  has  several  advantages  over  the  currently 
utilized  techniques  of  immunoprecipitation  and  blotting  from  sodium  dodecyl  sulfate  polyacrylamide 
gels  (Western  blots)  in  that  it  does  not  require  radioactive  labeling  of  antigen  and  that  it  avoids  dena- 
turation  of  proteins,  a  process  which  may  destroy  the  antigenic  site  recognized  by  a  monoclonal  antibody. 
The  existence  of  antigens  common  to  different  stages  of  the  parasite,  but  of  distinct  charge,  prompts 
further  investigation  as  to  the  nature  of  the  antigenic  site  (protein  or  sugar  determinant)  and  the  com- 
parison of  Eastern  blot  and  immunoprecipitation  results  by  two-dimensional  OTarrell  gels. 


PARASITOLOGY  AND  PATHOLOGY  393 

L.  enriettii  a-tubulin  is  produced  in  vivo  by  Escherichia  coli.  PAMELA  LANGER, 
MICHELE  JUNGERY,  AND  DYANN  WIRTH  (Harvard  University). 

The  study  of  parasites  at  the  molecular  level  is  limited  by  the  difficulty  of  isolation  and  character- 
ization of  parasite  proteins.  As  an  initial  attempt  to  get  expression  of  a  parasite  protein  in  bacteria,  we 
introduced  a  molecular  hybrid  of  genomic  L.  enriettii  a-tubulin  DNA  and  pBR322  (pLTl  constructed 
by  D.  Wirth)  into  maxicells.  We  report  here  that  we  have  been  able  to  detect  the  synthesis  of  Leishmania 
a-tubulin  in  E.  coli.  Lysates  of  maxicells  containing  pLTl  or  pBR322  were  spotted  onto  nitrocellulose 
filters  and  incubated  with  rabbit  anti-L.  enriettii  a-tubulin  antibody  and  I25l-labeled  protein  A.  Spots  of 
bacteria  containing  pBR322  showed  no  reaction  with  antibody  whereas  those  of  an  L.  enriettii  extract 
of  bacteria  containing  the  a-tubulin  gene  showed  a  strongly  positive  reaction.  The  synthesized  gene 
product  retained  its  antigenic  properties  to  the  extent  that  it  was  able  to  react  with  the  rabbit  anti-L. 
enriettii  a-tubulin  antibody. 

It  is  not  yet  known  whether  the  transcription  was  initiated  at  the  a-tubulin  gene  promoter  or  another 
site  such  as  the  pBR322  /3-lactamase  gene  promoter.  Further  studies  are  in  progress  to  determine  whether 
the  L.  enriettii  a-tubulin  gene  has  a  promoter  functional  in  bacteria.  Such  a  promoter  could  be  useful 
in  the  construction  of  cloning  vectors  for  other  parasite  proteins. 

In  summary,  we  have  observed  the  synthesis  in  bacteria  of  a  parasite  protein  which  retains  antigenic 
properties. 

A  phytomastigophorean  infection  of  embryonating  sea  hares  Aplysia  californica. 
Louis  LEIBOVITZ  (Laboratory  for  Marine  Animal  Health,  Marine  Biological 
Laboratory)  AND  THOMAS  R.  CAPO. 

A  specific  phytomastigophorean  infection  of  laboratory-cultured  embryonating  sea  hares  (Aplysia 
californica)  is  reported.  Earliest  microscopically  detectable  clinical  signs  were  observed  on  the  third  day 
after  egg  cases  were  laid.  It  could,  however,  be  observed  at  any  point  of  embryonal  development  after 
the  third  until  the  time  of  veliger  release,  usually  on  the  eighth  day.  Earliest  stage  of  infection  was  initiated 
by  rupture  of  clear  thin-walled  cysts,  20  to  40  nm  length  or  width,  discharging  small  infective  round  or 
oval  organisms,  3  to  6  ftm,  with  euglenoid-type  motility.  The  liberated  organisms  actively  penetrated  the 
outer  wall  of  the  egg  case,  forming  fistulous  tracts  and  becoming  attached  to  the  embryos  in  the  com- 
partments of  the  egg  case.  The  organisms  migrated  to  the  yolk  tissues  within  the  valve,  beginning  a  period 
of  feeding,  growth  and  reproduction.  The  resulting  mature  sausage-shaped  trophozoites,  15  to  40  nm, 
conformed  to  morphologic  description  of  phytomastigophora.  The  disease  spread  through  the  egg  mass 
by  direct  extension  resulting  in  embryo  erosion,  lysis,  and  ultimate  death.  Bacteria  and  ciliates  were  noted 
secondarily,  in  late  stages  of  the  disease.  If  infection  occurred  early  (3  days  after  egg  masses  were  laid), 
embryonal  mortality  was  high,  often  reaching  100  percent.  In  late  infection  (after  the  fifth  day),  mortality 
was  much  lower  (10  to  20  percent).  Surviving  larvae  remained  carriers  of  the  disease  organism.  Prevention, 
control,  and  eradication  of  the  disease  is  currently  being  studied. 

We  wish  to  thank  Ms.  Amy  Stone  and  Susan  L.  Perritt  for  technical  assistance.  This  project  has 
been  supported,  in  part,  by  grants  from  the  Division  of  Research  Resources,  National  Institutes  of  Health 
(1-40-PRO 1333-01)  and  the  National  Institutes  of  General  Medical  Science  (GM23540-06). 

A  competitive  inhibition  test  for  diagnosis  of  schistomiasis  using  monoclonal  anti- 
bodies. MARTIN  PAMMENTER  (South  African  Medical  Research  Council,  Box 
17120,  Congella  4013,  Natal,  Rep.  South  Africa),  PAUL  JOHNSON,  AND  DON 
HARN. 

Experiments  were  designed  to  test  the  serodiagnostic  potential  of  a  monoclonal  antibody  which 
binds  to  surface  membranes  of  the  helminth  parasite  Schistosoma  mansoni.  The  antibody  is  partially 
protective  in  passive  transfer  experiments  and  is  known  to  be  nonreactive  to  heterologous  (filarial)  hel- 
minth antigens. 

The  test  was  designed  as  a  competitive  inhibition  of  the  binding  of  the  monoclonal  antibody  to  S. 
mansoni  soluble  egg  extract  by  serum  from  infected  persons  using  a  solid  phase  ELISA.  5".  haematobium- 
infected  sera  were  obtained  from  schoolchildren  of  Kwa  Zulu  in  Southern  Africa  while  control  sera  were 
drawn  from  members  of  the  MBL. 

Initial  experiments  suggest  that  after  pre-incubation  with  a  1:16  dilution  of  infected  serum  there  is 
an  appreciable  reduction  in  binding  of  the  monoclonal  antibody  when  compared  to  controls.  This  in- 
hibitory activity  is  TCA-precipitable  and  can  also  be  precipitated  in  the  range  of  20%  to  40%  saturated 
ammonium  sulphate.  The  activity  can  also  be  at  least  partially  removed  by  passage  of  the  serum  through 
Protein  A-Sepharose. 


394  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

These  results  suggest:  1)  the  reaction  is  a  competitive  antibody  interaction,  2)  the  antigen  to  which 
the  monoclonal  antibody  is  directed  is  immunogenic  in  naturally  infected  people,  3)  the  antigen  is 
common  to  S.  mansoni  and  S.  haemalobium. 

(Martin  Pammenter  received  financial  aid  from  the  South  Africa  Medical  Research  Council  to  attend 
the  MBL  Biology  of  Parasitism  course.) 

Comparison  of  labeled  membrane  proteins  of  pathogenic  and  non-pathogenic  South 
American  trypanosomes.  DEBRA  ROWSE-EAGLE,  CARL  A.  BOSWELL,  TECIA 
ULISSES  DE  CARVALHO,  AND  MIERCIO  PEREIRA  (Tufts  University  Medical 
School). 

Trypanosoma  cruzi,  T.  rangeli  and  T.  conorhini  share  common  triatomine  vectors,  but  T.  cruzi  is 
pathogenic  for  man  whereas  T.  rangeli  and  T.  conorhini  are  not.  These  host  specificities  may  be  modulated 
by  the  presence  or  absence  of  unique  surface  membrane  components.  The  epimastigote  stage  of  these 
trypanosomatids  was  grown  in  liquid  culture  and  surface  labeled  with  I25I  using:  1 )  Bolton-Hunter  reagent, 
which  conjugates  to  amino  groups  via  an  active  ester  reaction;  2)  lodogen,  which  labels  exposed  tyrosine 
residues;  or  3)  5-iodo-l-napthylamine  (INA),  which  labels  hydrophobic  amino  acids  and  lipids. 

Labeling  with  lodogen  or  Bolton-Hunter  reagent  resulted  in  electrophoretic  patterns  which  were 
characteristic  for  each  species  of  trypanosome.  While  slight  differences  of  mobility  patterns  were  evident 
between  T.  rangeli  and  T.  conorhini,  there  were  major  differences  between  these  non-pathogens  and 
T.  cruzi. 

In  another  experiment  cells  were  treated  with  trypsin,  then  labeled  with  lodogen  to  see  if  cryptic 
membrane  components  were  exposed.  The  band  patterns  of  the  non-pathogenic  species  were  altered  very 
little,  but  major  changes  occurred  with  T.  cruzi.  Three  bands  (80,000,  52,00,  and  16,000  daltons)  dis- 
appeared and  were  replaced  by  a  single  new  band  (14,400  daltons). 

All  three  species  have  major  membrane  proteins  that  label  with  INA  and  migrate  on  8-20%  poly- 
acrylamide  gel  electrophoresis  at  molecular  weights  of  50,000,  35,000,  and  20,000  daltons. 

In  summary,  it  appears  that  membrane  proteins  accessible  to  surface  labeling  vary  between  species, 
but  integral  membrane  proteins  labeled  by  INA  are  highly  conserved. 

Membrane  labeling  of  protective  antigens  of  schistosomula  o/Schistosoma  mansoni. 

DAN  ZlLBERSTEIN,  PAUL  JOHNSON,  MlERCIO  PEREIRA  AND  DON  HARN  (Har- 
vard Medical  School). 

The  non-permeant  (125I)-iodogen  and  N-succinimidyl-3(4  hydroxy,5-('25I)iodophenyl)-propionate 
(Bolton-Hunter)  and  the  hydrophobic  5(125I)-iodonapthyl  azide  (INA)  were  used  to  surface  label  schis- 
tosomula of  Schistosoma  mansoni.  Using  these  three  reagents  nine  iodinated  proteins  (molecular  weights 
of  14,000,  20,000,  28,000,  35,000,  40,000,  50.000,  80,000,  94,000  and  125,000)  were  identified  by  SDS- 
PAGE.  An  additional  30,000  molecular  weight  component  was  labeled  by  both  INA  and  Bolton-Hunter 
reagents.  Immunoprecipitates  using  both  chronic  mouse  sera  and  monoclonal  antibody  contained  two 
proteins  (40,000  and  94,000  daltons)  which  have  been  labeled  by  both  INA  and  iodogen  reagents. 

Since  INA  reagent  labels  proteins  embedded  in  the  lipid  bilayer,  and  since  the  proteins  labeled  by 
this  reagent  were  also  labeled  by  the  reagents  that  label  proteins  externally  exposed  in  the  outer  membrane, 
the  INA  labeled  bands  are  integral  membrane  proteins.  Furthermore,  the  monoclonal  antibody  used  in 
this  study  was  shown  to  be  surface  membrane  specific.  This  observation  suggests  that  the  antigenic  epitope 
recognized  in  the  antigen  is  in  the  extra-membrane  portion  of  these  molecules. 


PHOTORECEPTORS 

Calcium  injections  increase  sensitivity  in  calcium  depleted  Limulus  ventral  photo- 
receptor  cells.  S.  R.  BOLSOVER  AND  J.  E.  BROWN  (State  University  of  New  York 
at  Stony  Brook). 

Bathing  Limulus  ventral  photoreceptors  in  low  calcium  sea  water  ([EGTA]  =  10  mA/,  free  [Ca] 
measured  to  be  2  X  10~6  M)  first  increased  the  light-induced  current  in  1-2  minutes.  After  15  minutes, 
there  was  a  progressive  decline  of  the  light-induced  current  and:  ( 1 )  an  increase  of  light  intensity  produced 
a  much  more  than  linearly  proportionate  increase  of  light-induced  current;  (2)  delayed,  apparently  re- 
generative currents  were  induced  by  long  flashes;  (3)  ionophoretic  injection  of  calcium  ions  from  an 


PHOTORECEPTORS  395 

intracellular  pipette  containing  Ca/EGTA  buffer  or  CaCl2  increased  the  light  response;  (4)  ionophoretic 
injection  of  EGTA  decreased  the  light  response.  When  cells  were  returned  to  ASW  (1CT2  M  Ca0)  their 
behavior  returned  to  normal.  To  monitor  changes  of  cytoplasmic  calcium  we  injected  single  cells  with 
aequorin.  Aequorin  luminescence  recorded  from  unilluminated  cells  fell  rapidly  in  low  Cao.  In  contrast, 
the  increase  of  luminescence  induced  by  a  bright  flash  (caused  by  a  light-induced  increase  of  cytoplasmic 
calcium)  did  not  change  significantly  during  the  first  several  minutes  in  low  Ca0;  however,  during  a 
prolonged  period  in  low  Cao  the  light-induced  increase  of  luminescence  declined  profoundly.  Returning 
the  cells  to  ASW  restored  both  the  resting  luminescence  and  the  light-induced  increase  of  luminescence. 
We  conclude  that  one  or  more  steps  in  the  transduction  system  in  Limuhts  photoreceptors  requires 
intracellular  calcium.  Bathing  in  low  Ca<,  for  prolonged  periods  reduces  both  the  free  cytoplasmic  calcium 
and  the  light-induced  release  of  calcium  from  internal  stores,  possibly  by  depleting  these  stores.  In  this 
condition,  we  propose  tentatively  that  light  induces  a  delayed  rise  of  cytoplasmic  calcium  that  acts  to 
increase  the  sensitivity  of  the  cell.  This  hypothesis  can  account  for  both  the  more  than  linear  stimulus- 
response  relation  and  the  delayed,  apparently  regenerative  light-induced  currents  recorded  in  voltage- 
clamped  cells  bathed  in  low  Cao. 

Supported  by  EY-01914  and  EY-01915. 

Nucleotide  injection  abolishes  the  discrete  waves  evoked  by  vanadate  in  Limulus 
photoreceptors.  D.  WESLEY  CORSON  AND  ALAN  FEIN  (Marine  Biological  Lab- 
oratory). 

In  previous  studies  of  Limulus  ventral  photoreceptors  we  have  reported  that  extracellular  application 
of  5  mM  vanadate  in  low  calcium  (1  mM)  artificial  sea  water  can  1)  induce  the  production  of  discrete 
waves  in  the  dark  and  2)  prolong  the  response  to  dim  flashes  of  light.  The  vanadate-induced  waves  were 
found  to  be  similar  to  those  normally  evoked  by  light.  Intracellular  injections  of  fluoride,  molybdate, 
tungstate,  or  GTP-7-S,  a  hydrolysis-resistant  analog  of  GTP,  have  previously  been  found  to  have  an 
effect  similar  to  that  of  vanadate,  while  previous  iontophoretic  injections  of  GTP  and  ATP  did  not  induce 
the  production  of  discrete  waves  or  prolong  the  light  response.  We  now  report  that  injection  of  either 
GTP  or  ATP  can  temporarily  abolish  both  the  discrete  waves  and  the  prolongation  of  the  light  response 
evoked  by  vanadate. 

GTP  (3  cells)  or  ATP  (5  cells)  was  pressure  injected  into  ventral  photoreceptors  from  electrodes 
containing  either  nucleotide  at  a  concentration  of  20  mM  along  with  80  mM  potassium  aspartate  (pH 
7.0).  Injection  of  either  of  the  nucleotides  abolishes  for  a  few  minutes  the  discrete  waves  evoked  by 
vanadate  in  the  dark.  Injection  of  either  of  the  nucleotides  also  temporarily  abolishes  the  vanadate- 
induced  prolongation  of  the  response  to  dim,  20-msec  flashes  but  does  not  appear  to  alter  the  response 
in  other  ways.  Control  injections  of  80  mM  KAsp  did  not  reverse  the  effects  of  vanadate  in  3  cells. 
Therefore  either  the  nucleotides  or  one  of  their  reaction  products  antagonize  both  of  the  effects  of 
vanadate. 

Supported  by  grants  from  the  NIH  and  the  Rowland  Foundation. 

Intracellular  injection  of  A  TP  can  reduce  spontaneous  discrete  wave  activity  in  Lim- 
ulus ventral  photoreceptors.  ALAN  FEIN  AND  D.  WESLEY  CORSON  (Marine  Bio- 
logical Laboratory). 

Spontaneous  activity  is  commonly  observed  throughout  the  nervous  system  in  the  absence  of  any 
apparent  stimulus.  In  Limulus  ventral  photoreceptors,  for  example,  spontaneous  discrete  waves  of  de- 
polarization occur  in  the  dark.  These  spontaneous  waves  are  very  similar  to  the  discrete  waves  that  are 
evoked  by  light  in  the  same  photoreceptors.  In  the  experiments  reported  here  ATP  was  injected  into 
ventral  photoreceptor  cells  by  applying  short  duration  pressure  pulses  to  the  back  of  intracellular  mi- 
cropipettes  containing  20  mM  Na2ATP,  100  mM  KAsp,  pH  7.0.  In  four  cells  we  found  that  the  rate  of 
spontaneous  wave  occurrence  was  reduced  following  the  injection  of  ATP.  For  the  two  most  active  cells 
having  spontaneous  rates  greater  than  1  per  sec  the  spontaneous  rate  fell  by  more  than  2-fold  following 
injection.  Injection  of  the  KAsp  solution  alone  did  not  lead  to  a  fall  in  the  rate  of  spontaneous  waves. 
The  ATP  induced  reduction  in  spontaneous  rate  occurred  without  any  apparent  change  in  the  efficacy 
with  which  light  could  induce  the  occurrence  of  discrete  waves  in  the  same  cells.  We  do  not  know  whether 
the  reduction  in  the  rate  of  spontaneous  waves  is  a  direct  effect  of  ATP  itself,  or  whether  the  ATP  enters 
into  a  series  of  reactions,  the  product  of  which  then  leads  to  a  fall  in  discrete  wave  rate.  The  spontaneous 
waves  of  ventral  photoreceptors  appear  to  arise  from  a  molecule  other  than  rhodopsin,  therefore  we 
suggest  that  the  effects  reported  here  do  not  reflect  an  effect  at  the  visual  pigment. 


396  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Photoreceptors  of  freshwater  turtles:  cell  types  and  visual  pigments.  LEO  E.  LIPETZ 
(The  Ohio  State  University)  AND  EDWARD  F.  MACNICHOL,  JR. 

Photoreceptors  of  three  species  of  freshwater  turtles  were  characterized  as  rod  or  double  or  single 
cone,  as  having  or  lacking  an  oil  droplet,  and  by  the  droplet's  spectral  transmission.  All  three  species  had 
rods  and  six  types  of  cones.  The  double  cone  consists  of  a  chief  cone  with  an  orange  (O)  droplet  and  a 
dropletless  accessory  cone.  The  N  droplet  has  no  absorption  in  the  visible  spectrum;  the  C  has  an 
absorption  peak  in  the  near  ultraviolet.  The  R,  O,  and  Y  droplets  transmit  significantly  only  at  wavelengths 
greater  than  about  580,  560,  and  525  nm,  respectively. 

The  droplets  and  visual  pigments  were  measured  with  a  computer-controlled,  photon-counting 
microspectrophotometer.  In  all  three  species  were  found  a  rod  visual  pigment  and  red-,  green-,  and  blue- 
sensitive  cone  pigments.  In  Chrysemys  scripta  elegans  for  each  visual  pigment  the  number  of  photore- 
ceptors  in  the  average,  the  mean  wavelength  of  maximum  absorption,  its  standard  deviation,  the  mean 
optical  density,  and  the  mean  half-width  were:  (a)  for  rod,  10,  519.4  ±  3.6  nm,  0.048,  148  TeraHertz 
(THz);  (b)  for  red-sensitive,  57,  622.7  ±  4.5  nm,  0.034,  142  THz;  (c)  for  green-sensitive,  24,  521.6  ±  3.1 
nm,  0.027,  134  THz;  (d)  for  blue-sensitive,  18,  461.6  ±  5.5  nm,  0.019,  133  THz.  For  Chrysemys  picta 
the  corresponding  values  were:  (a)  for  rods,  13,  521.3  ±  1.1  nm,  0.048,  123  THz;  (b)  for  red-sensitive, 
15,  623.6  ±  2.6  nm,  0.032,  127  THz;  for  green-sensitive,  8,  520.6  ±  2.9  nm,  0.025,  133  THz;  (d)  for 
blue-sensitive,  9,  461.1  ±  5.2  nm,  0.022,  133  THz. 

For  the  above  two  species  plus  Chelydra  serpentina  the  visual  pigment  was  identified  in  the  following 
total  numbers  of  each  type:  accessory  cones,  red,  134;  chief  cones,  red,  156;  R-cones,  red,  192,  and  green, 
2;  Y-cones,  green,  183;  C-cones,  blue,  125;  and  N-cones,  red,  25. 

This  work  was  supported  by  NEI  grants  EY  03743  and  EY  0239905. 

Evidence  for  the  release  of  a  catalytic  agent  during  the  latent  period  of  invertebrate 
phototransduction.  RICHARD  PAYNE  AND  ALAN  FEIN  (Marine  Biological  Lab- 
oratory). 

Dark-adapted  Limulus  ventral  photoreceptors  respond  to  a  dim  flash  with  a  latency  of  100  ms. 
Steady  background  illumination  decreases  the  latency  of  the  response  to  a  superimposed  flash  and  greatly 
reduces  the  response  amplitude.  The  decrease  in  latency  is  thought  to  be  due  to  the  release  by  the 
background  light  of  an  agent  that  increases  the  rate  of  an  early  reaction  in  phototransduction.  The  aim 
of  the  present  study  is  to  demonstrate  that  this  agent  is  released  during  the  latent  period  of  the  response 
of  a  dark-adapted  cell  to  a  bright  flash  and  to  determine  its  radius  of  diffusion. 

We  have  investigated  the  response  of  dark-adapted  cells  to  10-ms  flashes  delivered  as  10  ^m  spots 
of  light.  The  latency  of  the  response  per  effectively  absorbed  photon  falls  from  100  to  50  ms  as  the  density 
of  effective  photons  is  increased  from  1  to  300  per  nm2.  That  a  density  of  >  1  effective  photon  per  nm2 
should  initiate  the  decreased  latency  suggests  that  the  agent  responsible  is  able  to  diffuse  over  at  least  the 
length  of  a  microvillus  during  the  latent  period  of  the  response.  Comparison  of  the  latent  period  of  the 
response  to  a  10-20  nm  diameter  spot  with  that  to  a  diffuse  light  suggests  an  upper  limit  of  approximately 
10  ^m  for  the  diffusion  radius  of  the  agent. 

Intracellular  injection  of  calcium  is  known  to  reduce  the  latency  of  the  response.  Calcium  has  also 
been  shown  to  be  released  following  illumination.  If  calcium  is  the  agent  responsible  for  the  decrease  in 
latency  that  we  observe,  then  we  predict  a  significant  local  release  of  calcium  during  the  latent  period 
of  the  response  to  a  bright  flash  delivered  to  a  dark-adapted  cell. 

Evidence  for  postnatal  morphogenesis  of  skate  rods.  R.  BRUCE  SZAMIER  (Harvard 
University  Medical  School),  HARRIS  RIPPS,  AND  DOUGLAS  TAATJES 

Visual  function  in  skates  (Raja  erinacea  and  R.  oscellata)  is  subserved  solely  by  the  scotopic  (rod) 
mechanism,  and  the  visual  cells  of  this  elasmobranch  contain  only  one  type  of  photopigment,  namely 
rhodopsin.  However,  electrophysiological  studies  have  demonstrated  that  the  photoreceptors,  as  well  as 
second-  and  third-order  retinal  neurons,  are  responsive  to  incremental  light  flashes  presented  on  back- 
ground luminance  levels  that  extend  well  into  the  photopic  range.  In  addition,  our  histological  sections 
show  a  number  of  small,  proximally  placed,  cone-like  elements  within  the  photoreceptor  layer,  which 
possibly  represent  another  class  of  visual  cell.  Nevertheless,  our  results  suggest  otherwise. 

Ultrastructural  and  histochemical  studies  showed  that  the  membranous  discs  of  the  outer  segments 
of  these  cells  were  isolated  from  the  plasma  membrane,  and  that  their  synaptic  terminals  appeared 
immature,  unlike  those  usually  associated  with  cone  receptors.  In  addition,  the  pattern  of  incorporation 


PHOTORECEPTORS  397 

of  3H-fucose,  as  revealed  by  radioautography,  was  similar  for  both  the  rods  and  the  smaller  visual  cells; 
i.e.,  the  label  was  concentrated  along  the  basal  discs  of  the  outer  segment.  When  we  examined  the  disc 
shedding  behavior  of  the  visual  cells  in  skates  entrained  for  two  weeks  or  longer  to  a  12:12  lightrdark 
cycle,  enhanced  phagocytic  activity  was  seen  only  following  light  onset. 

The  middle  portion  of  the  inner  nuclear  layer  of  younger  animals  contained  large  numbers  of 
undifferentiated  cells  with  dense  nuclei  and  little  cytoplasm.  These  cells  decreased  in  number  with  age 
and  were  occasionally  seen  in  the  OPL  or  ONL.  3H-thymidine  autoradiography,  used  to  identify  prolif- 
erating cells,  revealed  that  these  undifferentiated  cells  were  post-mitotic  and  that  retinal  neurons  were 
being  formed  by  cell  division  only  in  a  circumferential  ring  at  the  outer  margin  of  the  retina. 

We  conclude  that  the  small  visual  cells  are  recently  differentiated  rods,  and  are  growing  and  being 
incorporated  into  the  photoreceptor  layer  of  the  retina.  These  rods  appear  to  originate  from  undiffer- 
entiated progenitor  cells  in  the  inner  nuclear  layer  which  migrate  to  the  outer  nuclear  layer. 

This  research  was  supported  by  grants  EY  02988  and  EY  00285  from  the  National  Eye  Institute, 
USPHS,  and  by  an  award  (to  D.T.)  from  Burroughs  Wellcome  Co.  to  Fight  For  Sight,  Inc.,  New  York. 

PHYSIOLOGY  AND  BIOPHYSICS 

Calcium-dependent  potassium  current  in  squid presynaptic  nerve  terminals.  GEORGE 
AUGUSTINE  AND  ROGER  ECKERT  (Dept.  of  Biology,  UCLA). 

In  a  previous  study  of  inactivation  of  calcium  current  in  squid  presynaptic  terminals  (Augustine  el 
a/.  1981,  Soc.  Neurosci.  Abstr.  7)  it  was  observed  that  the  Cd-sensitive  inward  current  elicited  by  de- 
polarizing voltage  clamp  pulses  relaxed  more  rapidly  than  the  Ca  conductance,  as  measured  by  Ca  tail 
currents.  We  report  here  that  this  discrepancy  is  due  to  the  presence  of  a  calcium-dependent  potassium 
current,  IK(Ca). 

Presynaptic  terminals  of  Loligo  pealei  were  voltage  clamped  with  the  3-microelectrode  method 
(Llinas  el  al.  1981,  Biophys.  J.  33:  289).  External  tetrodotoxin  and  3,4-diaminopyridine  and  internal 
tetraethylammonium  (TEA)  were  used  to  minimize  currents  flowing  through  sodium  and  delayed  rectifier 
channels.  Under  these  conditions  the  inward  current  elicited  by  100-300  msec  depolarizations  was  fol- 
lowed upon  repolarization  by  a  slow  outward  tail  current.  This  slow  tail  current  (r  =0. 1  sec)  was  blocked 
by  Cd,  had  a  reversal  potential  near  EK  (approx.  -80  mV),  and  was  sensitive  to  the  extracellular  con- 
centration of  K  ions.  Both  the  slow  tail  current  and  relaxation  on  inward  current  were  greatly  reduced 
by  external  TEA  (25-200  nuW).  These  features  are  characteristic  of  the  Ca-activated  K  current,  IK<ca), 
in  many  cells,  and  indicate  that  the  early  relaxation  of  inward  current  seen  without  extracellular  TEA 
primarily  reflects  the  simultaneous  activation  of  this  outward  current.  Decay  of  the  Ca  conductance, 
determined  from  tail  current  measurements,  is  a  slow,  exponential  process  with  a  time  constant  of  1.5 
to  5.3  s. 

Supported  by  the  Muscular  Dystrophy  Assn.  and  USPHS  NS  8364. 


Reproductive  strategies  of  bivalve  mollusks  from  deep-sea  hydrothermal  vents  and 
intertidal  sulfide-rich  environments.  CARL  J.  BERG,  JR.  (Marine  Biological  Lab- 
oratory) AND  PHILIP  ALATALO. 

A  comparison  of  clams  (Calyptogena  magnified)  from  deep-sea  hydrothermal  vent  areas  (2500  m) 
with  clams  (Codakia  orbicularis)  from  intertidal,  sulfide-rich  creeks  and  turtlegrass  beds  reveals  several 
similarities.  Individuals  of  both  species  are  thought  to  derive  major  portions  of  their  nutrients  from 
chemoautotrophic  bacteria  within  their  gills,  based  upon  morphological,  histological,  isotope,  and  enzyme 
analyses.  They  also  become  sexually  mature  at  a  relatively  young  age  and  release  gametes  over  prolonged 
periods  of  time.  Both  species  spawn  eggs  which  are  enclosed  in  gelatinous  capsules.  Eggs  of  C.  orbicularis 
are  108-1 12  /um  in  diameter  and  are  surrounded  by  a  350  urn  diameter  capsule.  Preserved  eggs  and 
capsules  of  C.  magnified  are  larger,  measuring  approximately  400  ^m  and  495  ^m  diameter,  respectively. 
Under  laboratory  conditions,  veliger  larvae  of  C.  orbicularis  hatch  from  individual  capsules  in  2-3  days 
and  are  planktonic  for  approximately  1 2  days.  Larvae  maintained  in  either  1  ^m  filtered  sea  water,  or 
sea  water  with  suspensions  of  cultured  phytoplankton,  undergo  metamorphosis  13-16  days  after  fertil- 
ization. Although  egg  size  is  greater  in  C.  magnified,  we  hypothesize  that  similar  development  occurs. 
Lecithotrophic  nutrition  of  these  larvae  may  be  supplemented  by  planktotrophic  and/or  chemoauto- 
trophic capabilities. 


ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

Mechanism  and  function  of  synchronous  flash  ing  in  the  firefly  Photinus  pyralis.  JOHN 
BUCK  (National  Institutes  of  Health),  FRANK  E.  HANSON,  ELISABETH  BUCK, 
AND  JAMES  F.  CASE. 

The  flying  male  emits  a  single  flash  about  every  6  s  at  23°C.  The  perched  female  responds  about 
2  s  after  each  male  flash.  The  male  reaches  the  female  via  a  succession  of  such  alternated  signals.  After 
one  male  initiates  dialogue  with  a  female,  other  males  often  join,  flashing  synchronously  with  the  original 
male.  Synchronization  occurs  when  male  A's  flash  impinges  on  B  1.5  s  or  less  before  B  is  due  to  flash. 
B's  flash  is  then  triggered  prematurely  and  his  flash-timing  and  -perceiving  cycle  is  reset.  In  pacemaker 
resetting  in  an  Oriental  species  flashing  is  delayed  one  full  timing  cycle  (Buck  el  a/.,  1981,  /.  Comp. 
Physiol.  144:  287).  In  contrast,  the  P.  pyralis  flash  delay  is  only  350  ms,  about  the  latency  of  cephalic 
electrical  stimulation,  as  if  the  timing  cycle  were  reset  to  its  end  rather  than  its  start. 

If  A  is  in  dialogue  with  a  female  when  he  resets  B,  B  can  then  respond  directly  to  A's  female  and 
compete  on  an  even  footing.  In  a  pair  interaction  A  thus  halves  his  own  chances  of  being  accepted  by 
the  female.  The  evolutionary  selection  of  male-male  flash-triggering  (synchrony)  therefore  seems  puzzling. 
In  larger  groups,  however,  flash  synchronization  could  prove  adaptive.  If  a  6th  male  joins  5  synchronized 
suitors  he  decreases  the  reproductive  prospects  of  each  of  the  5  from  1/5  to  1/6  but  increases  his  own 
prospects  (with  that  female)  from  zero  to  1/6.  He  gains  no  individual  advantage  over  any  other  male  but 
wins  a  chance  to  compete  for  a  rarely  found  prize.  During  the  many  random  group  encounters  of  one 
male  with  others  during  his  lifetime  these  (strongly  advantageous)  opportunities  to  join  courtships  of 
other  males  more  than  offset  the  equal  number  of  (weakly  disadvantageous)  occasions  when  he  inad- 
vertently causes  another  male  to  joint  his  own  courtship. 

A  single  calcium-mediated  process  can  account  for  both  rapid  and  slow  phases  of 
inactivation  exhibited  by  a  single  calcium  conductance.  ROGER  ECKERT,  DOUG- 
LAS EWALD,  AND  JOHN  CHAD  (Department  of  Biology,  UCLA,  Los  Angeles). 

Cells  L2-L6  in  Aplysia  calijornica  were  voltage  clamped  in  artificial  sea  water  containing  0.45  mM 
tetrodotoxin,  200  mM  tetraethylammonium  chloride,  and  5  mM  4-amino-pyridine  to  isolate  the  Ca 
current,  IQ,-  Depolarizations  were  to  0  mV  or  less,  lasting  up  to  900  ms.  Calcium  tail  currents  measured 
at  EK  were  proportional  to  the  inward  current  at  all  times,  indicating  an  absence  of  contamination  by 
K  current.  Inactivation  kinetics,  determined  from  computer  fits,  were  correlated  closely  with  current 
strength  (i.e.  peak  ICa),  and  were  only  secondarily  influenced  by  membrane  voltage,  Vm.  Thus,  when  ICa, 
elicited  at  0  mV,  was  progressively  reduced  with  extracellular  Cd2+,  the  inactivation  kinetics  slowed 
dramatically,  approximately  matching  those  of  currents  of  similar  peak  ICa  elicited  by  smaller  depolar- 
izations before  the  Cd  block.  Inactivation  occurred  with  two  exponential  phases,  a  rapid  rhl,  and  a  slower 
Th2  asymptotic  to  a  noninactivating  component,  1-, .  Progressive  reduction  of  ICa  by  whatever  means 
resulted  in  a  progressive  disproportionate  loss  of  the  rM  component,  and  a  slowing  of  rh2.  At  small 
currents  only  rh2  remained.  Furthermore,  injection  of  EGTA  slowed  both  rhl  and  rh2,  and  increased  1^. 
These  findings  indicate  that  Th,  and  rh2  both  reflect  Ca-dependent  processes. 

These  kinetics,  along  with  other  features  of  the  calcium  current,  were  simulated  by  iterative  solution 
of  the  following  equation,  based  on  Hodgkin-Huxley  m2  activation  kinetics  plus  Ca-mediated  inactivation 
proportional  to  intracellular  free  Ca2+: 

lea  =  [GCa(Vm  -  ECa)][m^  -  (m,,  -  m0)e  l/Tm]2-  1/(1  +  K-S) 

in  which  K  =  efficacy  of  Ca2+  in  inactivating  Ca  channels;  S  =  Jo  (1  -  B)ICa  dt;  and  B  =  probability  that 
free  Ca2+  at  membrane  inner  surface  will  be  lost  to  diffusion  or  buffering.  The  model  simulates  the 
biexponential  kinetics  of  calcium  inactivation  seen  in  molluscan  neurons,  although  it  contains  only  a 
single  class  of  channels  and  includes  no  voltage-dependent  inactivation.  The  biexponential  kinetics  arise 
from  the  interplay  of  m2  activation  and  Ca-mediated  inactivation  that  is  proportional  to  current-depen- 
dent accumulation  of  Ca2+. 

Supported  by  USPHS  NS8364  and  NSF  BNS  80-12346. 

Incorporation  of  a  calcium-selective  conductance  from  Paramecium  cilia  in  a  planar 
lipid  bilayer.  B.  E.  EHRLICH  (Albert  Einstein  College  of  Medicine),  A.  FINKEL- 
STEIN,  M.  FORTE,  C.  KUNG. 

Paramecium  is  well  suited  to  reconstitution  studies  of  the  voltage-dependent  Ca++  channel  for  four 
reasons:  1)  the  ionic  currents  have  been  well  studied  electrophysiologically,  2)  behavioral  mutants  lacking 
Ca++  currents  are  available,  3)  voltage-dependent  Ca++  channels  exist  on  the  cilia  only,  and  4)  large 


PHYSIOLOGY  AND  BIOPHYSICS  399 

quantities  of  ciliary  membrane  vesicles  (CMV)  can  be  prepared.  When  CMV  are  incorporated  into  a 
planar  lipid  bilayer  in  the  presence  of  KC1,  very  large  (100-400  pS),  very  slow  (open  tens  of  seconds), 
voltage-dependent  cation-selective  "channels"  are  seen.  We  think  these  records  represent  the  large  pores 
found  after  EDTA  treatment  of  intact  Paramecium.  Addition  of  100  nM  Ca++  to  the  vesicle-containing 
bath  irreversibly  inhibits  75-90%  of  the  membrane  conductance.  To  investigate  the  properties  of  the 
remaining  10-25%  of  the  conductance,  incorporation  with  Ca++  as  the  only  permeant  cation  has  been 
done.  When  a  Ca++  gradient  is  imposed,  a  current  is  measured.  From  biionic  potentials,  the  relative 
permeability  of  Ca++:Sr++:Ba++:Mg++  is  1:1:0.5:<0.01.  Symmetric  addition  of  K+  at  20-100  times  the 
Ca++  concentration  will  shunt  the  Ca++  current.  This  result  suggests  that  there  is  a  parallel  pathway  for 
monovalent  cations. 

Initial  tests  with  metal  blockers  show  that  half  the  current  is  inhibited  by  1.5  mM  cobalt,  0.5  mA/ 
cadmium,  or  10  \iM  lanthanum.  These  values  are  consistent  with  those  from  intact  preparations.  When 
CMV  from  pawn  mutants  (Paramecium  with  ~  10%  of  the  Ca++  conductance  of  wild  type  cells)  are 
incorporated  into  the  bilayer,  we  see  the  same  degree  of  background  conductance.  However,  for  a  given 
Ca++  gradient  the  Ca++  current  is  one-tenth  the  current  obtained  with  wild  type  CMV. 

In  summary,  the  reconstituted  currents  are  comparable  to  in  situ  Ca++  currents  in  ionic  selectivity, 
in  degree  of  block  by  metals,  and  by  lack  of  response  in  one  mutant.  We  are  now  investigating  the 
voltage-dependence  of  these  currents. 

Supported  by  NIH  grant  GM  29210-05,  NSF  grant  BNS-79 18554,  and  a  Muscular  Dystrophy 
Foundation  Postdoctoral  Fellowship  to  BEE. 

Transduction  and  voltage-dependent  currents  ofstatocyst  hair  cells  in  Hermissenda. 
JOSEPH  FARLEY  AND  DANIEL  L.  ALKON  (Section  on  Neural  Systems,  Lab.  of 
Biophysics,  NINCDS,  NIH,  MBL). 

Statocyst  hair  cells  in  the  mollusk  Hermissenda  process  gravitational  information.  Motile  cilia  trans- 
duce the  effects  of  gravity  through  active  interaction  with  statoconia,  resulting  in  mechanical  deformation 
of  the  hair  cell  membrane  at  the  basal  insertion  region  of  the  axoneme.  Such  stimulation  produces 
increased  voltage  noise  and  a  depolarizing  generator  potential  if  sufficiently  intense.  We  have  studied  the 
processes  of  integration  and  amplification  of  these  sensory  signals  in  hair  cell  somatic  membrane  through 
current-noise  analysis  of  resting  potential  conductances,  and  voltage-clamp  studies  of  the  voltage-depen- 
dent conductances. 

Current-noise  amplitude  was  1-2  orders  of  magnitude  greater  for  loaded  vs  unloaded  hair  cells,  and 
progressively  increased  with  holding  potentials  more  negative  than  -40  mV.  Removal  of  extracelluar 
Na+  from  the  bath  provided  a  clear  and  reversible  decrease  in  noise  amplitude.  These  observations 
indicate  that  Na+  ions  contribute  greatly  to  the  depolarizing  voltage  noise  in  the  undamped  cell. 

We  have  identified  two  voltage-dependent  K+  currents  in  the  hair  cells.  The  fast,  rapidly  inactivating 
current  (IA)  is  elicited  at  -30mV,  is  TEA-resistant,  is  abolished  by  4-aminopyridine  (4-AP),  and  is 
inactivated  by  prior  depolarization.  The  slower,  sustained  K+  current  (IB)  is  selectively  reduced  by  TEA, 
but  not  4-AP.  Both  currents  are  calcium  regulated.  Removal  of  extracellular  Ca++  increased  IA  by  10- 
35%  in  10  of  1 1  cells  studied;  IB  was  affected  to  a  much  smaller  degree. 

Despite  the  fact  that  the  transduction  portion  of  Hermissenda  photoreceptors  and  hair  cells  are 
derived  from  quite  different  membrane  types  (rhabdomeric  and  ciliary,  respectively),  the  method  of 
sensory  encoding,  integration,  and  transmission  of  electrical  signals  is  remarkably  similar.  In  both  cases, 
signal  detection  is  accomplished  by  increases  in  voltage  noise  arising  from  inward  Na+  (and  also  Ca++) 
current  which  summates  to  yield  a  depolarizing  generator  potential.  The  IA  and  IB  currents  appear  to  be 
identical,  with  the  former  regulated  by  Ca++. 

Electrochemical,  electron  spin  resonance  and  spectroscopic  measurements  of  some 
cytotoxic  quinones.  PETER  R.  C.  GASCOYNE,  JANE  A.  MCLAUGHLIN,  RONALD 
PETHIG,  AND  ALBERT  SZENT-GYORGYI  (Marine  Biological  Laboratory). 

Cosgrove  el  al.  (1952,  J.  Chem.  Soc.  4821-4823)  have  isolated  methoxy-p-benzoquinone  and  2,6- 
dimethoxy-p-benzoquinone  from  fermented  wheat  germ,  and  Jones  et  al.  (1981,  J.  Natural  Prod.  44: 
493_494)  have  found  the  2,6-dimethoxyquinone  to  be  cytotoxic. 

Studies  in  this  laboratory  on  mice  inoculated  with  Ehrlich  ascites  have  indicated  that  the  combination 
of  ascorbic  acid  (AA)  with  2,5-  or  2,6-dimethoxyquinone  exhibits  strong  cytotoxic  properties.  The  com- 
bination of  AA  with  benzoquinone,  monomethoxyquinone  or  2,3-dimethoxyquinone  does  not  exhibit 
such  cytotoxicity.  None  of  the  quinones  were  found  to  exhibit  such  cytotoxicity  in  the  absence  of  AA. 
To  understand  the  basic  chemical  properties  that  could  be  responsible  for  these  observations,  time- 
resolved  electron  spin  resonance  measurements  have  been  made  of  the  reactions  between  AA  and  these 
various  quinones.  For  the  2,5-  and  2,6-dimethoxyquinone,  evidence  is  found  for  the  production  of  short- 


400  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

lived  ascorbate  free  radicals  which  are  then  scavenged  by  the  quinone  to  form  long-lived  semiquinone 
radicals.  Such  effects  were  not  observed  for  the  monomethoxy-,  2,3-dimethoxy-,  and  benzo-quinone 
interactions  with  AA. 

We  have  determined  the  electrochemical  potentials  for  the  various  quinone-hydroquinone  redox 
couples,  and  the  redox  potentials  (at  pH  7.4  and  25°C.)  for  2,5-  and  2,6-dimethoxyquinone  (35.2  and 
79.2  mV,  respectively)  lie  close  to  that  for  the  dehydroascorbate-ascorbate  couple  (46.6  mV).  For  meth- 
oxyquinone,  2,3-dimethoxyquinone  and  benzoquinone  the  corresponding  potentials  were  found  to  be 
1 64,  185,  and  262  m  V,  respectively.  These  data,  together  with  spectroscopic  and  electrochemical  titrations, 
provides  support  for  the  viewpoint  that  the  cytotoxic  properties  of  the  2,5-  and  2,6-dimethoxyquinone 
are  related  to  the  production  of  relatively  long-lived  free  radicals  as  a  result  of  one-  rather  than  of  two- 
electron  reductions  by  the  ascorbic  acid. 

Professor  Gabor  Fodor  kindly  prepared  the  quinones.  This  work  is  supported  by  the  National 
Foundation  for  Cancer  Research. 

Incorporation  of32P-phosphate  into  lipids  and  proteins  by  intact  squid  giant  axons. 
R.  M.  GOULD  (Inst.  for  Basic  Research,  Staten  Island),  C.  A.  MANCUSO,  P. 
GALLANT,  AND  I.  TASAKJ. 

Axon  processes,  though  lacking  the  capacity  for  protein  translation,  contain  enzymes  both  for  syn- 
thesis and  metabolism  of  lipids  and  modification  of  proteins.  The  localization  of  these  latter  activities 
to  axons  has  been  demonstrated  biochemically  with  pure  axoplasm,  extruded  from  squid  giant  axons. 
We  have  demonstrated  that  axoplasm  catalyzes  the  incorporation  of  12P-phosphate  into  both  lipids  and 
proteins.  A  variety  of  other  potential  lipid  precursors,  including  myoinositol,  choline,  glycerol,  serine  and 
glucosamine  are  also  used  in  phospholipid  and/or  glycolipid  synthesis  by  extruded  axoplasm. 

To  study  the  relationship  of  axonal  lipid  and  protein  metabolism  to  active  properties  of  the  axon, 
we  felt  it  would  be  necessary  to  use  an  intact  axon  preparation.  Intact  axons,  incubated  in  sea  water 
containing  radioactive  precursors,  incorporate  label  into  lipids  and  proteins.  However,  when  the  axoplasm 
and  Schwann  cell-rich  sheath  are  separated  by  extrusion  of  axoplasm  and  analyzed  separately,  the  sheath 
always  contained  several  times  more  labeled  lipid  and/or  protein,  independent  of  the  precursor.  In 
contrast,  when  we  injected  32P-phosphate  into  the  axons,  the  precursor  was  avidly  retained  in  the  axoplasm 
and  the  labeling  of  axoplasmic  lipids  and  proteins  exceeded  that  of  the  sheath. 

We  have  studied  the  incorporation  of  injected  32P-phosphate  into  lipids  and  proteins  of  intact  giant 
axons  under  four  conditions,  1)  unstimulated  (resting),  2)  electrically  stimulated  at  60/sec,  3)  TEA  in  the 
injection  solution,  and  4)  NaCl  in  the  injection  solution.  There  were  significant  increases  in  the  incor- 
poration of  label  into  lipids  of  both  axoplasm  and  sheath  (includes  both  axolemma  and  Schwann  cells) 
in  stimulated,  as  well  as  TEA-  and  NaCl-injected  axons  compared  with  unstimulated  controls.  The 
increases  in  axoplasmic  labeling  were  most  apparent  in  the  inositol  lipids,  particularly  the  polyphos- 
phoinositides.  There  were  some  indications  that  protein  phosphorylation  was  increased  by  the  presence 
of  TEA.  These  results  show  that  we  have  a  method  for  studying  phospholipid  and  post-translational 
protein  metabolism  in  the  squid  giant  axon  and  that  this  metabolism  (at  least  with  32P-phosphate)  is 
responsive  to  physiological  stimuli. 

Supported  by  grant  NS  12980  from  NIH. 

Isolation  of  an  extreme  clump-forming  bacterium.  ROBERT  R.  HALL  (Nantucket 
High  School,  Nantucket,  MA  02554),  H.  O.  HALVORSON,  AND  K.  KEYNAN. 

While  flocculation  of  microorganisms  into  aggregates  is  frequently  observed  in  natural  environments, 
the  mechanisms  regulating  flocculation  are  not  always  well  understood.  To  study  this  process  we  isolated 
pure  cultures  of  flocculating  cells  from  a  waste  treatment  plant.  Microscopic  examination  of  the  floes 
indicate  that  they  contain  a  wide  diversity  of  types  with  a  rod-like  organism  being  the  predominant  type. 
Two  observations  provided  the  basis  for  its  isolation.  First  the  clumps  grow  rapidly  at  low  temperature 
in  a  dilute  synthetic  medium.  Secondly  when  the  clumps  are  sonicated  in  EDTA  the  cells  are  briefly 
dispersed  yielding  motile  cells  which  rapidly  clump.  Pure  cultures  were  eventually  obtained  by  isolating 
colonies  rich  in  clumping  cells,  sonicating  these  in  EDTA,  rapidly  diluting  and  plating  these  on  solid 
medium  and  finally  selecting  the  small,  late-forming  colonies  arising  from  single  cells.  This  process  was 
repeated  until  pure  cultures,  as  judged  by  microscopy  and  colony  forms,  were  obtained.  The  final  isolated 
culture  is  a  gram-negative  rod  which  exists  free  as  a  motile  organism,  then  attaches  to  glass  or  forms  a 
tightly  packed  aggregate.  Tests  with  n-heptane  show  that  flocculation  may  be  due  to  cell  surface  hydro- 
phobicity. 


PHYSIOLOGY  AND  BIOPHYSICS  401 

Characterization  of  a  detoxifying  enzyme  from  squid  salivary  gland  by  use  ofSoman, 
DFP,  and  manganous  ion.  FRANCIS  C.  G.  HOSKIN  (Illinois  Institute  of  Tech- 
nology) AND  ROBERT  D.  PRUSCH. 

Although  an  enzyme  that  hydrolyzes  the  cholinesterase  inhibitor  diisopropyl  phosphorofluoridate 
(DFP)  is  present  in  mammalian  (e.g.,  rat)  kidney,  another  DFPase  with  a  different  molecular  weight, 
structure,  and  properties  is  present  in  squid  (Loligo  pealei)  nerve  (see  Hoskin  and  Roush  1982,  Science 
215:  1255-1257  for  earlier  references).  Squid  nerve  DFPase  detoxifies  DFP  more  rapidly  than  another 
organophosphorus  compound,  ethyl  N,N-dimethylphosphoramidocyanidate  (Tabun),  whereas  this  order 
is  reversed  for  mammalian  kidney  DFPase.  This  criterion  is  cumbersome:  DFP  releases  two  strong  acids 
whereas  Tabun  releases  one,  making  the  pH-stat  method  ambiguous;  DFP  releases  fluoride  whereas 
Tabun  does  not,  making  the  sensitive  fluoride  electrode  method  impossible.  Parallel  research  on  venomous 
neurotoxic  agents  unexpectedly  revealed  a  high  level  of  DFPase  in  squid  posterior  salivary  gland.  We 
report  a  new  criterion  for  differentiating  the  two  DFPases,  and  its  application  to  the  squid  salivary  DFPase. 
Another  organophosphorus  compound,  1,2,2,-trimethylpropyl  methylphosphonofluoridate  (Soman)  is 
hydrolyzed  20-40  times  faster  than  DFP  by  rat  kidney,  whereas  squid  nerve  DFPase  hydrolyzes  DFP 
5-10  times  faster  than  Soman,  all  under  comparable  conditions.  Rat  kidney  DFPase  is  stimulated  2-  to 
3-fold  by  4  X  10~4  A/  Mn++,  whereas  squid  nerve  DFPase  is  unaffected  or  slightly  inhibited.  These  ob- 
servations form  the  basis  for  distinguishing  squid  nerve  DFPase  from  mammalian  kidney  DFPase,  the 
names  not  being  rigorously  indicative  of  enzyme  source  or  substrate.  On  this  basis  the  DFPase  found 
in  squid  saliva  is  identifiable  as  squid  type  DFPase.  The  enzyme  is  different  from  the  proteinous  toxin 
also  found  in  squid  saliva.  There  is  nearly  twice  as  much  DFPase  in  female  squid  saliva  as  in  male  saliva. 
The  enzyme  is  also  present  in  whole  salivary  gland.  The  natural  substrate  and  physiological  role  for  this 
enzyme,  or  for  the  superficially  similar  enzyme  in  mammalian  kidney,  is  the  subject  of  continuing 
research. 

Supported  by  an  ARO  grant. 

Selection  and  properties  of  glucose  transport  mutants  of  Vibrio  parahaemolyticus. 
H.  L.  KORNBERG  (Department  of  Biochemistry,  University  of  Cambridge,  Cam- 
bridge CB2  1QW,  U.  K.),  T.  M.  PERNACK,  AND  D.  J.  SCHNELL. 

Like  Escherichia  coli,  Vibrio  parahaemolyticus  takes  up  glucose  via  the  phosphoenolpyruvate-de- 
pendent  phosphotransferase  (PT)  system;  unlike  E.  coli,  it  can  apparently  carry  out  cation-linked  glucose 
transport  since  5  \iM  carbonylcyanide  m-chlorophenylhydrazone  (CCCP)  powerfully  inhibits  it.  In  order 
to  determine  the  physiological  role  of  these  systems,  mutants  resistant  to  inhibition  by  non-catabolizable 
glucose  analogs  were  selected  (a)  by  cycling  V.  parahaemolyticus  repeatedly  through  media  containing 
fructose  ±  methyl  a-D-glucoside;  (b)  by  repeated  culture  of  the  Vibrio  on  a  mixture  of  0.5%  peptone, 
0.3%  yeast  extract,  and  0.4%  methyl  a-D-glucoside  (Matsumoto  el  al.  1974,  J.  Bad.  119:  632-634);  and 
(c)  by  culturing  the  organism  on  L-lactate  in  the  presence  of  5-thio-D-glucose.  Mutants  thus  selected  did 
not  grow  on  glucose  as  sole  carbon  source,  did  not  take  up  14C-labeled  glucose  or  methyl  a-D-glucoside 
and,  when  rendered  permeable  with  toluene,  did  not  effect  the  phosphoenolpyruvate-dependent  phos- 
phorylation  of  these  hexoses;  however,  the  mutants  were  unimpaired  in  the  uptake  and  utilization  of 
mannose,  glucoasmine,  and  fructose.  These  latter  three  hexoses  are  known  to  share  with  glucose  the 
PtsM  uptake  system  of  E.  coli,  which  also  effects  the  uptake  of  2-deoxyglucose.  Since  this  glucose  analog 
was  taken  up  by  V.  parahaemolyticus  to  only  a  negligible  extent,  and  since  the  mutants  took  up  and 
phosphorylated  mannose  without  significant  glucose  transport,  the  PtsM  system  of  the  Vibrio  can  play 
at  best  only  a  minor  role  in  glucose  transport.  Similarly,  since  the  mutants  that  lacked  the  ability  to 
phosphorylate  methyl  a-D-glucoside  also  lacked  the  ability  to  take  up  more  than  traces  of  glucose,  the 
cation-linked  glucose  transport  system  (if,  indeed  it  is  present)  cannot  be  involved  in  glucose  uptake  to 
any  major  extent. 

Characterization  of  D-xylose  and  D-glucose  transport  systems  in  Spirochaeta  au- 
rantia.  CYNTHIA  A.  PADEN,  SUSAN  ROBERTS,  AND  E.  P.  GREENBERG  (Cornell 
University,  Ithaca,  NY). 

Transport  of  14C-D-glucose  and  14C-D-xylose  by  the  gram-negative  bacterium,  Spirochaeta  aurantia 
M 1 ,  was  investigated.  For  these  studies,  cells  were  suspended  in  10  mM  potassium  phosphate  buffer,  pH 
7,  to  a  final  density  of  approximately  2  X  108  cells/ml.  Uptake  of  both  sugars  was  linear  over  the  4  to 
5  minute  duration  of  the  experiments.  A  kinetic  analysis  for  uptake  indicated  apparent  Km  values  for 
D-xylose  and  D-glucose  of  7  and  4  nM,  respectively.  The  apparent  Vmax  for  D-xylose  uptake  was  0.25 


402  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

nmoles/min/108  cells  and  the  apparent  Vmax  for  D-glucose  uptake  was  0.5  nmoles/min/ 108  cells.  Transport 
of  both  substrates  was  completely  inhibited  when  cells  were  suspended  in  10  mM  potassium  arsenate 
rather  than  potassium  phosphate.  Addition  of  PMS  and  ascorbate  provided  cells  in  potassium  arsenate 
with  a  proton  motive  force,  but  did  not  reverse  the  inhibition  of  D-xylose  or  D-glucose  uptake.  Uptake 
of  both  sugars  was  inhibited  by  less  than  25%  in  the  presence  of  carbonyl  cyanide  m-chlorophenylhy- 
drazone  (2  p.M),  an  agent  to  collapse  the  proton  motive  force  in  S1.  aurantia.  An  osmotic  shock  decreased 
D-glucose  uptake  from  0.43  to  0.04  nmoles/min/108  cells  and  D-xylose  uptake  from  0.24  to  0.05  nmoles/ 
min/108  cells.  The  velocity  of  D-glucose  transport  was  not  decreased  in  the  presence  of  1  mM  D-mannose, 
D-allose,  a-methyl-D-glucoside,  or  3-O-methyl-D-glucose  and  was  decreased  by  1 5%  in  the  presence  of 
2  mM  D-xylose.  D-xylose  transport  was  not  decreased  in  the  presence  of  1  mM  2-O-methyl-D-xylose, 
a-methyl-D-xyloside,  /8-methyl-D-xyloside,  or  a-methyl-D-glucoside,  but  was  decreased  85%  in  the  pres- 
ence of  10  nM  D-glucose.  These  studies  indicate  specific  uptake  systems  for  D-glucose  and  D-xylose. 
Both  systems  require  a  high-energy  phosphorylated  compound  for  transport  rather  than  a  proton  motive 
force.  Furthermore,  the  uptake  systems  are  sensitive  to  osmotic  shock.  These  features  are  similar  to 
those  of  the  binding  protein-mediated  transport  systems  in  other  gram-negative  bacteria  such  as 
Escherichia  coli. 

Parts  of  this  research  were  supported  by  the  Foundation  for  Microbiology  and  NASA  NAGW-306. 

An  endopeptidase  inhibitor,  similar  to  vertebrate  a-2  macroglobulin,  present  in  the 
plasma  of  Limulus  polyphemus.  JAMES  P.  QUIGLEY  (Marine  Biological  Labo- 
ratory), PETER  B.  ARMSTRONG,  PAUL  GALLANT,  FRED  R.  RICKLES  AND  WAL- 
TER TROLL. 

The  plasma  of  vertebrates  contains  a  variety  of  macromolecular  inhibitors  of  proteolytic  enzymes 
whose  function  is  to  bind  and  inhibit  proteases  of  both  endogenous  and  exogenous  origin.  One  important 
member  of  this  family  of  plasma  proteins  is  a-2  macroglobulin  (a2M).  This  protease  inhibitor  is  effective 
against  a  wide  spectrum  of  endopeptidases  and  acts  by  forming  a  complex  that  shields  the  active  site  of 
the  protease  from  macromolecular  substrates  but  leaves  the  enzyme  free  to  hydrolyze  low  molecular 
weight  substrates.  The  inhibitory  activity  of  a2M  is  sensitive  to  mild  acidification  and  also  methylamine 
treatment  since  the  complex  formed  between  a2M  and  protease  is  stabilized  by  thiol  ester  bonds. 

A  potent  protease  inhibitory  activity  has  now  been  detected  in  the  hemolymph  of  the  horseshoe 
crab  Limulus  polyphemus  and  possesses  many  of  the  characteristics  of  vertebrate  a2M.  Hemolymph  was 
prepared  by  bleeding  pre-chilled  crabs  under  sterile,  endotoxin-free  conditions.  Cells  were  removed  im- 
mediately by  low  speed  centrifugation.  Hemocyanin  was  removed  from  the  plasma  by  centrifugation  at 
100,000  x  g  for  4  hours.  The  resulting  clear  supernatant  contained  only  2-4%  of  the  plasma  protein  and 
most  of  the  protease  inhibitory  activity.  The  inhibitory  activity  in  the  supernatant  was  characterized 
using  a  number  of  protease  assays  including  the  hydrolysis  of  l4C-casein,  125I-fibrin  and  arginyl  p  ni- 
troanilide. 

The  supernatant  inhibited  the  activity  of  trypsin,  chymotrypsin,  plasmin  and  elastase.  The  inhibitory 
activity  was  due  to  a  high  molecular  protein  which  was  shown  to  be  sensitive  to  mild  acidification  and 
methylamine  treatment.  The  activity  of  trypsin  against  low  molecular  weight  substrates  was  not  inhibited 
by  the  supernatant,  indicating  that  the  active  site  of  the  protease  remains  free. 

To  our  knowledge  this  represents  the  first  demonstration  of  a  plasma  protease  inhibitor  in  Limulus. 
The  striking  similarity  of  the  inhibitor  to  vertebrate  a2M,  coupled  with  the  fossil  record  of  Limulus, 
suggests  that  such  protease  inhibitors  are  relatively  ancient  molecules.  The  function  and  pathophysiology 
of  the  molecule  in  the  horseshoe  crab  is  now  under  investigation. 

Electrogenic  Na+/K  +  pump  current  and  flux  measurements  on  voltage-clamped,  in- 
ternally dialyzed  squid  axons.  R.  F.  RAKOWSKI  AND  PAUL  DE  WEER 
(Washington  University,  School  of  Medicine,  St.  Louis). 

An  improved  "pump-clamp"  technique  has  been  developed  for  the  direct  measurement  of  electro- 
genie  Na+/K+  pump  current  and  isotopic  fluxes  in  internally  dialyzed  squid  giant  axons.  A  stable,  low- 
noise  voltage-clamp  circuit  is  used  to  maintain  the  membrane  potential  to  within  ±40  ^V.  The  electrogenic 
pump  current  is  measured  as  the  change  in  holding  current  produced  upon  addition  of  ouabain  or  the 
reversible  cardiotonic  steroid  dihydrodigitoxigenin  (H2DTG)  to  the  sea  water  bathing  the  central  pool 
of  the  experimental  chamber.  This  central  pool  is  isolated  from  the  adjacent  end-pools  by  petroleum 
jelly  seals.  The  magnitude  of  the  pump  current  was  about  1  /^A/cm2.  The  method  assumes  that  the  toxins 
H2DTG  and  ouabain  produce  no  change  of  passive  membrane  conductance  or  equilibrium  potential  of 
any  permeant  ion.  This  assumption  is  validated  by  the  absence  of  a  response  to  toxin  addition  when 
pump  operation  was  stopped  by  elimination  of  a  required  ion  or  substrate.  That  is,  no  change  in  holding 


PHYSIOLOGY  AND  BIOPHYSICS  403 

current  was  produced  by  toxin  addition  if  Na+,  K+,  Mg2+  or  nucleotides  were  eliminated  from  the  internal 
and  external  solutions.  The  elimination  of  both  internal  Na+  and  external  K+  produced  reversal  of 
electrogenic  pump  current  and  Na+  flux.  Both  forward  and  reverse  pump  currents  were  inhibited  by 
membrane  hyperpolarization,  suggesting  that  the  pumping  rate  is  not  necessarily  proportional  to  the 
thermodynamic  driving  force.  Preliminary  data  suggest  that  the  stoichiometry  of  the  squid  axon  Na+/K+ 
pump  is  2Na+/lK+. 

Supported  by  NIH  grants  NS-11223,  NS- 14856,  and  the  Muscular  Dystrophy  Association  of 
America. 

Effect  of  methyl  a-D-glucoside  on  the  growth  of  enteric  bacteria:  inhibition  and  escape 
from  inhibition.  D.  J.  SCHNELL  (Department  of  Life  Sciences,  University  of 
Nebraska,  Lincoln,  NE  68508),  T.  M.  PERNACK,  AND  H.  L.  KORNBERG. 

When  cultures  of  enteric  bacteria,  grown  on  fructose,  are  diluted  into  fresh  fructose  medium  that 
also  contains  methyl  a-D-glucoside,  further  growth  is  inhibited.  However,  in  medium  of  low  phosphate 
content  (<0.6  mA/),  this  stasis  is  overcome  within  2-4  h:  the  organisms  "escape"  from  inhibition  and 
their  subsequent  growth  is  not  affected  by  the  glucose  analog. 

We  have  measured  the  uptake  of  '4C-labeled  carbohydrates  by  cells  grown  on  a  variety  of  carbo- 
hydrates, and  also  the  phosphoenolpyruvate-dependent  phosphorylation  of  these  carbohydrates  ("PT- 
activity")  by  cells  rendered  permeable  with  toluene.  Vibrio  parahae molyticus,  like  Escherichia  coli,  effects 
concomitantly  the  uptake  and  phosphorylation  of  methyl  a-D-glucoside  via  the  PT-system  (Km  s»  10 
nAf).  Cells  that  are  at  the  point  of  "escaping"  from  inhibition  by  2  mM  methyl  a-D-glucoside  contain 
in  undiminished  activity  the  PT-components  that  were  present  at  the  time  the  glucose  analog  was  added; 
however,  they  have  elaborated  an  intracellular  activity  that  causes  methyl-a-D-['4C]glucoside  taken  up 
to  be  rapidly  lost  again  from  the  cells.  It  is  possible  that  exposure  of  the  culture  to  glucose  analogs,  in 
media  containing  low  phosphate  concentrations,  favors  induction  of  an  intracellular  phosphatase  as  well 
as  the  periplasmic  alkaline  phosphatase  known  to  be  formed. 

After  "escape,"  the  glucose-specific  components  of  the  PT-system  cease  to  be  further  made  during 
subsequent  growth  in  fructose  in  the  presence  of  methyl  a-D-glucoside,  though  the  fructose  PT-activity 
continues  to  be  synthesized.  Since  glucose  induces  the  glucose-specific  PT  system,  its  repression  suggests 
that  methyl-tt-D-glucoside  phosphate  prevents  further  expression  of  the  appropriate  genes. 

Perfusion  of  the  squid  stellate  ganglion  through  its  blood  supply:  implications  for 
morphological  and  physiological  studies  of  the  squid  giant  synapse.  E.  F.  STAN- 
LEY (Johns  Hopkins  Medical  School)  AND  W.  J.  ADELMAN,  JR. 

The  synapse  between  2nd  and  3rd  order  giant  axons  in  the  squid  stellate  ganglion  has  been  used 
to  examine  many  aspects  of  synaptic  transmission.  However,  one  of  the  difficulties  in  using  this  prep- 
aration for  both  physiological  and  morphological  studies  is  the  considerable  diffusion  barrier  between 
bathing  medium  and  synapse.  To  circumvent  this  problem  we  have  perfused  the  ganglion  through  its 
arterial  blood  supply. 

The  anterior  aorta  was  cannulated  distal  to  the  single  branch  that  bifurcates  to  supply  both  stellate 
ganglia,  in  squid  ranging  from  55  to  115  mm  mantle  length.  The  aorta  was  tied  off  proximal  to  this 
branch  and  the  arteries  leading  to  the  ganglia  were  tied  off  proximal  to  the  right  ganglion  and  just  distal 
to  the  left  ganglion.  The  left  ganglion  could  now  be  perfused  in  situ  or  after  removal  in  vitro.  Efficacy 
of  perfusion  was  tested  by  passing  dye  or  fixative  through  the  ganglion  and  by  testing  time  taken  for 
LaCl3  to  block  transmission.  Infusion  of  dye  or  fixative  resulted  in  a  virtually  immediate  color  change 
of  the  whole  ganglion  and  the  adjoining  nerves  which  was  most  evident  around  the  cell  bodies  but  was 
also  evident  in  small  vessels  within  the  neuropil.  Application  of  LaCl3  (which  is  believed  to  block  Ca2+ 
influx  into  the  pre-synaptic  terminal  and  hence  transmitter  release)  to  the  bath  blocked  transmission  in 
38  minutes,  confirming  the  high  diffusion  barrier.  Infusion  of  the  same  LaCl3  solution  blocked  trans- 
mission in  33  or  63  seconds  (two  experiments).  We  conclude  that  perfusion  through  the  blood  supply 
greatly  improves  access  of  substances  to  the  giant  synapse. 

Comparative  microbiology  of  metal  surfaces  in  sea  water.  MARIANNE  WALCH,  PAUL 
J.  BOYLE,  AND  RALPH  MITCHELL  (Laboratory  of  Microbial  Ecology,  Division 
of  Applied  Sciences,  Harvard  University,  Cambridge,  MA  02138). 

Metal  surfaces  in  aquatic  systems  are  sites  of  intense  microbial  activity,  which  can  result  in  the 
enhancement  of  corrosion  processes.  We  conducted  experiments  to  understand  the  processes  involved 
in  the  attachment  of  various  marine  bacteria  to  specific  metals  and  alloys  commonly  used  in  ocean 


404  ABSTRACTS  FROM  MBL  GENERAL  MEETINGS 

engineering  applications.  Four  types  of  metal — 316  stainless  steel,  titanium,  90-10  copper-nickel  (CA 
706),  and  aluminum  bronze  D  (CA  614) — were  exposed  both  to  pure  cultures  of  marine  bacteria  and, 
in  situ,  to  sea  water  in  Eel  Pond  at  Woods  Hole,  Massachusetts.  After  exposure  for  varying  time  periods 
the  metals  were  removed  and  examined  using  acridine  orange  direct  counts  and  scanning  electron  mi- 
croscopy. 

Present  data  indicate  that  both  qualitative  and  quantitative  differences  in  the  attached  microbial 
communities  occur  in  response  to  different  types  of  metal  surfaces.  In  general,  the  two  copper  alloys 
supported  smaller  and  less  active  populations  of  bacteria  than  did  similarly  treated  stainless  steel  and 
titanium.  Removal  of  the  protective  oxide  film  from  a  metal  surface  by  acid  pickling  or  sanding  appeared 
to  alter  bacterial  attachment  and  growth,  at  least  in  the  short  term.  Also,  bacteria  grew  more  rapidly  and 
reached  higher  surface  populations  when  metals  were  exposed  previously  to  high  concentrations  of  dis- 
solved organic  matter.  Dramatic  differences  in  bacterial  attachment  behavior  were  seen  between  exper- 
iments run  in  artificial  sea  water,  in  natural  sea  water  in  vitro,  and  in  natural  sea  water  in  situ,  emphasizing 
the  need  to  examine  further  the  effect  of  environment  on  microbial  attachment  to  metals. 

This  research  was  supported  by  Office  of  Naval  Research  contract  number  N00014-81-K-0624.  The 
technical  assistance  of  Susan  Wolff  is  gratefully  acknowledged. 

Effects  of  H2O2  on  the  dogfish  (Mustelus  canisj  ocular  lens.  SEYMOUR  ZIGMAN, 
TERESA  PAXHIA,  BLENDA  ANTONELLIS,  AND  WILLIAM  WALDRON  (University 
of  Rochester  School  of  Medicine,  Rochester,  NY  14642). 

High  levels  of  the  strong  oxidant  H2O2  (10  6-10  4  M)  were  found  in  human  aqueous  humors  by 
Garner  and  Spector  (1981).  In  this  study,  fresh  dogfish  lenses  were  incubated  in  elasmobranch  Ringer's 
media  plus  ascorbic  acid  at  2.4  mg/ml  and  concentrations  of  H2O2  from  10"  '  M  to  10  6  M  for  up  to  72 
h  at  20-22°C.  In  some  experiments,  they  were  also  exposed  to  a  long  wavelength  UV  emitting  lamp 
(5  mW/cm2  at  365  nm),  with  suitable  dark  controls  in  parallel.  Incubated  lenses  developed  cortical 
opalescence  in  4  h  and  dense  opacity  in  17  h  due  to  H2O2  at  10  4  M  (minimal)  to  10  '  M  (maximal) 
concentrations.  Histological  examination  revealed  no  structural  defects  in  the  outer  cortex  or  lens  epi- 
thelium. Lenses  did  not  swell,  nor  was  X6Rb  or  14C-«-amino  isobutyric  acid  uptake  inhibited  by  10~2  M 
H2O2  for  44  h.  Starch  iodide  and  dichlorophenyl  indophenol  assays  showed  only  1  and  4%  of  the  [H2O2] 
in  the  medium  was  present  in  the  lens  cortex  and  epithelium.  Proteins  of  incubated  lenses  were  extracted 
and  separated  by  homogenization.  centrifugation,  and  polyacrylamide  gel  electrophoresis  analysis.  Ag- 
gregation via  -SS-  bonds  was  found  to  be  stimulated  by  H2O2  at  10  3  M,  but  not  at  10~5  M.  Additional 
near-UV  light  exposure  enhanced  aggregation;  catalase  and  DTT  inhibited  it  strongly.  Optical  spectros- 
copy  of  lens  proteins  and  free  tryptophan  showed  that  10  4  M  H2O2  plus  near-UV  light  destroyed  their 
280  nm  absorption  and  stimulated  fluorescence  with  excitation  at  360  nm  and  emission  at  440  nm.  Such 
emissions  are  found  in  the  proteins  of  aging,  near-UV  exposed,  and  brown  cataractous  human  lenses. 
Thus,  if  [H2O2]  in  aqueous  humor  is  >10  4  M,  lens  proteins  may  be  aggregated  to  form  lens  opacities, 
but  uptake  of  water,  salt,  and  amino  acids  is  not  altered  appreciably. 

Support:  NEl;  RPB;  Mullie  Fund;  Pledger  Fund. 


Continued  from  Cover  Two 

^  ,? 


with  their  headings  and  footnotes,  should  be  typed  on  separate  sheets,  numbered  with  consecutive  Roman 
numerals,  and  placed  after  the  Literature  Cited.  Figure  legends  should  contain  enough  information  to 
make  the  figure  intelligible  separate  from  the  text.  Legends  should  be  typed  double  spaced,  with  con- 
secutive Arabic  numbers,  on  a  separate  sheet  at  the  end  of  the  paper.  Footnotes  should  be  limited  to 
authors'  current  addresses,  acknowledgments  or  contribution  numbers,  and  explanation  of  unusual  ab- 
breviations. All  such  footnotes  should  appear  on  the  title  page.  Footnotes  are  not  normally  permitted 
in  the  body  of  the  text. 

4.  A  condensed  title  or  running  head  of  no  more  than  35  letters  and  spaces  should  appear  at  the  top 
of  the  title  page. 

5.  Literature  cited.     In  the  text,  literature  should  be  cited  by  the  Harvard  system,  with  papers  by 
more  than  two  authors  cited  as  Jones  el  al.,  1980.  Personal  communications  and  material  in  preparation 
or  in  press  should  be  cited  in  the  text  only,  with  author's  initials  and  institutions,  unless  the  material  has 
been  formally  accepted  and  a  volume  number  can  be  supplied.  The  list  of  references  following  the  text 
should  be  headed  LITERATURE  CITED,  and  must  be  typed  double  spaced  on  separate  pages,  con- 
forming in  punctuation  and  arrangement  to  the  style  of  recent  issues  of  The  Biological  Bulletin.  Citations 
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(i.e.  J.  Cancer  Res.) 

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time  for  corrections  to  these  (other  than  corrections  of  printers'  or  editors'  errors). 


CONTENTS 


CHRISTY,  JOHN  H. 

Adaptive  significance  of  semilunar  cycles  of  larval  release  in  fiddler 
crabs  (genus  Uca):  test  of  an  hypothesis    \/.-.  -. 251 


EMLET,  RICHARD  B. 

Echinoderm  calcite:  a  mechanical  analysis  from  larval  spicules    264 


ESCALONA  DE  MOTTA,  GLADYS,  DAVID  S.  SMITH,  MARILYN  CAYER,  AND 

JOSE  DEL  CASTILLO 

Mechanism  of  the  excitation-contraction  uncoupling  of  frog  skeletal 
muscle  by  formamide   .  .  .  f>vv» :'.,.'•,.<? 276 


FORWARD,  R.  B.,  JR.,  K.  LOHMANN,  AND  T.  W.  CRONIN 

Rhythms  in  larval  release  by  an  estuarine  crab  (Rhithropanopeus  har- 
risii )   ,     Y/v-  •  ''•*  •  ••  ••'•'.  • •/•  .-.'.-^'. . .  .^ 287 


HOPKINS,  PENNY  M. 

Growth  and  regeneration  patterns  in  the  fiddler  crab,  Uca  pugilator   301 


MARTIN,  VICKI  J.,  AND  FU-SHIANG  CHIA 

Fine  structure  of  a  scyphozoan  planula,  Cassiopeia  xamachana 320 


O'CONNOR,  KATHLEEN,  PHILIP  J.  STEPHENS,  AND  JOHN  M.  LEFEROVICH 
Regional  distribution  of  muscle  fiber  types  in  the  asymmetric  claws  of 
Californian  snapping  shrimp  ..•.-% 329 


SCHUEL,  HERBERT,  PRAMILA  DANDEKAR,  AND  REGINA  SCHUEL 

Urea  parthenogenetically  activates  the  cortical  reaction  and  elongation 

of  microvilli  in  eggs  of  the  sea  urchin,  Strongylocentrotus  purpuratus    337 


WATTS,  STEPHEN  A.,  R.  E.  SCHEIBLING,  ADAM  G.  MARSH,  AND  JAMES  B. 

McCLINTOCK 

Effect  of  temperature  and  salinity  on  larval  development  of  sibling  spe- 
cies  of  Echinaster  (Echinodermata:   Asteroidea)  and   their   hybrids    348 


ABSTRACTS  OF  PAPERS  PRESENTED  AT  THE  GENERAL  SCIENTIFIC  MEETINGS 

OF  THE  MARINE  BIOLOGICAL  LABORATORY 

Actin,  microtubules,  etc,    . : i  \ .  ^. , . '.>  . « ;1 . , . . ; /. .•* 355 

Ecology    ,^w  .;> . . '.-(. 362 

Fertilization  and  development , .  .\.^ . t ^  -  371 

Neurobiology    .  .7^. >  .  - 379 

Parasitology  and  pathology V  .j>. ' 391 

Photoreceptors ^\.  :/&^.  i. 394 

Physiology  and  biophysics    . . .  0)\. .  \;. .. ... ,~. 397 


Volume  163  ,  Number  3 


THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 


Editorial  Board 

DANIEL  L.  ALKON,  National  Institutes  of  Health  and     MICHAEL  G.  O'RAND,  Laboratories  for  Cell  Biology, 

Marine  Biological  Laboratory  University  of  North  Carolina  at  Chapel  Hill 

ROBERT  B.  BARLOW,  JR.,  Syracuse  University  RALPH  S.  QUATRANO,  Oregon  State  University  at 

Corvallis 
State  University  of  New  York     JQEL  L  RoSENBAUM)  Yale  University 

DAV.D  H.  EVANS,  University  of  Florida  DOROTHY  M.  SKINNER,  Oak  Ridge  National 

Laboratory 

HARLYN  O.  HALVORSON,  Brandeis  University  JOHN  D.  STRANDBERG,  Johns  Hopkins  University 

V) 

SAMUEL  S.  KOIDE,  The  Population  Council,  J-  RICHARD  WHITTAKER,  Boston  University, 

Rockefeller  University        Marine  Program  and  Marine  Biological  Laboratory 

E.  O.  WILSON,  Harvard  University 
FRANK  J.  LONGO,  University  of  Iowa 

GEORGE  M.  WOODWELL,  Ecosystems  Center,  Marine 
GEORGE  O.  MACKIE,  University  of  Victoria  Biological  Laboratory 

. 

Editor:  CHARLES  B.  METZ,  University  of  Miami 

' 


DECEMBER,  1982 


Printed  and  Issued  by 
LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 
LANCASTER,  PA. 


THE  BIOLOGICAL  BULLETIN 

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Continued  on  Cover  Three 


Reference:  Biol.  Bull.  163:  405-419.  (December  1982) 


INVERTEBRATE  CELL  VOLUME  CONTROL  MECHANISMS:  A 

COORDINATED  USE  OF  INTRACELLULAR  AMINO  ACIDS 

AND  INORGANIC  IONS  AS  OSMOTIC  SOLUTE 

SIDNEY   K.   PIERCE 

Department  of  Zoology,  University  of  Maryland,  College  Park,  MD  20742, 
and  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

ABSTRACT 

All  cells  have  some  capacity  for  cell  volume  regulation  when  confronted  with 
a  hypoosmotic  stress.  The  basis  of  this  physiological  response  is  an  extrusion  of 
intracellular  osmotic  solute.  The  cells  of  euryhaline  osmoconforming  invertebrates 
are  capable  of  regulating  volume  over  a  wide  range  of  external  osmotic  concentra- 
tions. Most  of  the  existing  data  indicate  that  these  cells  utilize  free  amino  acids  from 
a  substantial  intracellular  pool  as  the  solute  source.  However,  recent  studies  indicate 
that  these  invertebrate  cells  utilize  inorganic  ions  as  osmotic  solute  as  well.  The 
relative  contribution  of  each  solute  type  varies  from  species  to  species  and,  perhaps, 
from  cell  type  to  cell  type.  The  two  solute  types  are  regulated  by  different  mechanisms 
and  often  with  different  time  courses,  but  both  solute  control  systems  function  in 
a  coordinated  manner  to  regulate  cell  volume.  In  addition,  evidence  is  appearing 
demonstrating  a  role  for  organic  solutes  in  the  volume  regulatory  processes  of  ver- 
tebrate cells.  At  present,  it  seems  that  the  volume  regulatory  mechanisms  utilized 
by  all  cells  may  be  more  similar  than  currently  thought,  differing  in  relative  con- 
tributions of  the  two  solute  types  rather  than  kind  of  solute  utilized. 

INTRODUCTION 

The  literature  reporting  on  studies  of  osmotic  control  amongst  the  invertebrates 
is  vast.  The  water  balance  mechanisms  utilized  by  invertebrate  species  have  been 
under  some  form  of  investigation  for  all  of  this  century  and  even  earlier.  In  company 
with  most  trends  in  research  of  biological  function,  water  balance  studies  have 
proceeded  from  analysis  of  whole  organism  responses  to  the  intricacies  of  cellular 
physiology.  The  studies  cited  in  the  following  pages  do  not  constitute  an  encyclopedic 
review,  but  rather  point  out  some  of  the  more  recently  discovered  features  of  in- 
vertebrate cellular  water  balance  systems  and,  where  there  are  some  data,  the  sim- 
ilarities of  the  cellular  osmotic  control  mechanisms  between  species,  the  presence 
or  absence  of  a  backbone  notwithstanding.  My  remarks  here  are  confined  only  to 
the  responses  to  hypoosmotic  stress  since  that  has  been  the  most  intensely  studied. 

Whole  animal  responses  —  some  generalities 

It  is  now  apparent  that  few,  if  any,  invertebrates  are  isosmotic  with  their  envi- 
ronment. Even  the  body  fluids  of  marine  osmoconformers  are  slightly  hyperosmotic 
to  their  environment  (Remmert,  1969;  Pierce,  1970;  Oglesby,  1978,  1981,  for  ex- 
amples). Thus,  most,  if  not  all,  animals  have  an  osmotic  gradient  between  the 
environment  and  the  extracellular  fluid  and  some  physiological  capacity  for  handling 

Received  9  July  1982;  accepted  24  September  1982. 

405 


406 


SIDNEY   K.   PIERCE 


the  water  movement  resulting  from  that  gradient.  This  form  of  osmotic  stress  is 
usually  a  modest  one  in  an  osmoconformer  and,  as  Oglesby  points  out  in  his  review 
(1981),  easily  handled  by  excretory  systems.  The  osmoregulators  may  have  much 
larger  osmotic  gradients  between  extracellular  fluids  and  the  environment,  but  in 
the  adapted  state  the  water  movements  are  dealt  with  by  appropriate  ionic  transport 
systems  of  excretory  systems,  gills,  guts,  and  integument.  Of  greater  physiological 
consequence  are  changes  in  the  osmotic  concentration  of  the  external  environment, 
for  example  an  alteration  in  salinity,  or  the  occurrence  of  some  pathology  causing 
malfunction  of  the  extracellular  osmotic  and  ionic  homeostatic  mechanisms.  The 
physiological  response  by  invertebrates  to  salinity  change  has  been  studied  in  great 
detail.  Pathological  studies  have  been  done  only  in  higher  vertebrates  (see  Pollock 
and  Arieff,  1980,  for  a  review)  for  the  most  part,  and  that  aspect  will  be  touched 
on  only  briefly  below. 

No  animal  appropriately  tested  behaves  like  a  piece  of  dialysis  tubing  when 
exposed  to  a  hypoosmotic  stress.  The  capacity  for  volume  control  under  such  a 
stress  may  be  limited  and  the  range  of  tolerated  osmotic  concentrations  narrow,  but 
some  volume  regulatory  capacity  is  present  nonetheless.  Osmoconforming  animals 
rapidly  swell  in  response  to  the  osmotic  influx  of  water  produced  by  the  hypoosmotic 


.  80 


26C 


A  10%  sw 

•  25%  sw 

50%sw 

75%sw 


10        12  24 

Time  (hr) 


36 


72 


FIGURE  1 .  The  pattern  of  whole  animal  volume  regulation  in  Elysia  chlorotica.  At  time  0,  animals 
acclimated  to  100%  sea  water  were  transferred  to  the  salinities  indicated  and  weighed  at  intervals  for  3 
days.  Each  point  is  a  mean  from  6  animals.  Error  bars  indicate  S.E.M.  (From  Pierce  et  al,  1982). 


INVERTEBRATE  CELL  VOLUME  CONTROL 


407 


stress,  but  with  time  in  the  reduced  salinity  will  at  least  partially  recover  the  original 
volume  (Fig.  1 ).  The  recovery  time  course  varies  from  species  to  species.  In  general, 
the  more  euryhaline  an  animal  the  more  rapid  the  recovery.  Osmoregulators  may 
show  a  similar  response  or  may  simply  swell  less  than  predicted  (see  Oglesby,  1981, 
for  a  thorough  review).  These  whole  animal  responses  are  the  result  of  water  balance 
mechanisms  which  function  at  two  levels  within  an  organism.  First,  the  extracellular 
systems  mentioned  above,  bulk  movement  of  extracellular  water  by  the  excretory 
system,  ion  transport  by  various  epithelia,  and  integumental  water  permeability  all 
function  in  some  combination  to  remove  the  excess  water  (again,  see  Oglesby,  1981). 
In  addition,  the  osmotic  influx  of  water  into  the  extracellular  compartments  results, 
perforce,  in  a  dilution  of  the  extracellular  environment.  This  dilution  places  an 
osmotic  stress  on  the  cells.  Thus,  although  it  can  not  be  distinguished  by  whole 
animal  measurements,  the  second  level  of  response  is  at  each  cell. 

All  cells  tested  to  date  have  some  volume  regulatory  ability.  Like  the  whole 
animal,  when  the  isolated  cell  is  exposed  to  a  hypoosmotic  stress,  it  swells.  With 
time  in  the  reduced  osmotic  concentration  the  cell  returns  toward  its  original  volume 
(Fig.  2a,  b).  Few  cells,  if  any,  are  able  to  recover  the  exact  original  volume.  Rather, 
an  incomplete  volume  regulation  is  the  rule.  The  cells  of  euryhaline  osmoconforming 
invertebrates,  often  naturally  exposed  several  times  daily  to  wide  and  rapid  osmotic 
fluctuations,  are  excellent  volume  regulators,  but  the  cells  from  invertebrate  species 
have  no  monopoly  on  this  response.  Vertebrate  cells  also  regulate  volume  albeit 
usually  over  comparatively  narrow  ranges  of  osmotic  concentration  (mammalian 


e 
_3 

"o 

r=      130 

o 

"o 


O 


120- 


110- 


100- 


A 


30  60  90 

Time(min) 


120 


I300-, 


1200 


£       1100- 


cn    1000- 

c 
o 

J= 

0  900 

d> 

1  800 


=       700 

<D 
O 

600 

100 
0- 


996"592  mosm 
996 ••  996  mosm 


B 


10        20         30        40        50 

Time  (min) 


60 


120        130 


FIGURE  2.  (A)  Pattern  of  volume  regulation  by  red  blood  cells  isolated  from  Noetia  ponderosa 
adapted  to  full  strength  sea  water  and  exposed  to  50%  sea  water  at  time  0.  Cell  volume  was  determined 
as  packed  cell  hematocrits.  (Data  from  Amende  and  Pierce,  1980).  (B)  Pattern  of  volume  regulation  by 
red  coelomocytes  isolated  from  Glycera  dibranchiata  adapted  to  996  mosm  and  then  exposed  to  the 
osmotic  concentration  indicated  at  time  0.  Cell  volume  was  measured  with  a  Coulter  counter  (Data  from 
Costa  et  al,  1980). 


408  SIDNEY   K.   PIERCE 

and  avian  red  blood  cells  [Kregenow,  1971;  Poznansky  and  Solomon,  1 972;  Schmidt 
and  McManus,  1974],  Ehrlich  ascites  cells  [Hendil  and  Hoffman,  1974;  Hoffman, 
1978],  mammalian  heart  [Thurston  el  ai,  1981],  mammalian  brain  [Pollock  and 
Arieff,  1980;  Thurston  el  aL  1980],  flounder  red  cells  [Fugelli,  1967;  Cala,  1977], 
Amphiuma  blood  cells  [Cala.  1980],  human  lymphocytes  [Bui  and  Wiley,  1981], 
and  rat  liver  [van  Rossum  and  Russo,  1981]  have  all  been  looked  at  in  this  regard). 
No  doubt  a  major  reason  for  the  weaker  volume  regulatory  ability  of  these  types 
of  cells  is  the  evolution  of  vertebrate  homeostatic  mechanisms. 

The  volume  regulating  capacity  of  the  cells  of  euryhaline  osmoconforming  in- 
vertebrates has  attracted  some  experimental  attention  from  investigators  with  one 
of  two  points  of  view  over  the  past  decade.  First,  from  the  point  of  view  of  envi- 
ronmental physiology,  these  cells  have  been  utilized  in  various  attempts  to  under- 
stand the  basis  of  salinity  tolerance.  Second,  from  the  standpoint  of  cellular  phys- 
iology, the  mechanisms  of  cell  volume  regulation  should  be  more  obvious  in  a  cell 
type  that  functions  over  a  wide  range  of  osmotic  concentrations  since  the  responses 
should  be  magnified.  The  utility  of  this  last  approach  is  only  of  value  if  all  cells  use 
a  mechanism  of  hydration  control  in  common,  at  least  in  its  general  characteristics. 
As  the  studies  of  invertebrate  cell  volume  regulation  unfolded,  similarities  with 
vertebrate  cell  mechanisms  were  indeed  found,  but  also  some  major  differences. 
More  recent  data  indicate  that  the  mechanisms  may  be  more  similar  than  we  first 
thought. 

Invertebrate  cell  volume  regulation  in  response  to  hypoosmotic  stress  -  -  the  early 
results 

The  general  features  of  cell  volume  regulation  are  the  same  regardless  of  the 
specific  source  of  the  cell  type.  A  reduction  in  external  osmotic  concentration  pro- 
duces cellular  swelling  due  to  osmotic  influx  of  water.  To  counter  the  swelling  and 
prevent  osmotic  lysis,  the  cells  expel  osmotic  solute  together  with  osmotically  ob- 
ligated water  and  cell  volume  recovers  back  toward,  but  usually  not  reaching,  the 
original  level.  Historically,  the  osmotic  solute  source  utilized  during  this  process  by 
marine  invertebrate  cells  was  presented  as  being  small  organic  molecules,  usually 
free  amino  acids,  occasionally  quaternary  ammonium  compounds.  Vertebrate  cells 
(more  likely,  terrestrial  animals)  or  cells  from  freshwater  invertebrates,  on  the  other 
hand,  usually  seemed  to  use  inorganic  ions  as  osmotic  solute.  Indeed  there  is  con- 
siderable evidence  in  support  of  this  dichotomy.  Each  species  seems  to  have  its  own 
unique  extracellular  osmotic  concentration,  but  in  general  the  bloods  of  marine 
animals  are  very  close  to  sea  water  in  osmotic  concentration  (900-1000  mosm), 
while  the  fluids  of  terrestrial  and  freshwater  animals  are  much  lower  in  concentra- 
tion. The  osmotic  equilibrium  between  the  cells  of  these  organisms  and  the  respective 
extracellular  fluids  is  such  that  the  osmotic  gradient  is  minimized  (although  not 
zero).  Thus,  the  cells  of  marine  invertebrates  presumably  have  osmotic  concentra- 
tions approximately  that  of  sea  water,  while  the  intracellular  osmotic  concentrations 
of  terrestrial  and  freshwater  animals  are  much  lower.  The  total  intracellular  inorganic 
ion  composition  of  vertebrates  seems  to  account  for  60-70%  of  the  intracellular 
osmotic  concentration  (see  the  reviews  of  Conway,  1957,  and  Burton,  1968,  and 
the  data  in  Prosser,  1973).  On  the  other  hand,  in  marine  invertebrates  the  intra- 
cellular inorganic  ion  composition  is  only  slightly  higher  than  vertebrate  levels 
(again,  see  Prosser's  [1973]  tables).  Thus,  the  total  inorganic  concentration  inside 
invertebrate  cells  is  much  lower  than  extracellular  concentrations.  The  physiological 
reason  for  this  ionic  discrepancy  is  not  clear.  There  seems  to  be  some  deleterious 


INVERTEBRATE  CELL   VOLUME  CONTROL  409 

sensitivity  of  some  enzymes  to  salt  concentrations  higher  than  those  found  inside 
cells  of  animals  adapted  to  sea  water  (Clark  and  Zounes,  1977;  Bowlus  and  Somero, 
1979;  Yancey  el  al.,  1982),  but  the  data  are  limited  at  present.  In  any  case,  the 
osmotic  differential  between  the  extracellular  and  intracellular  fluids  in  marine  in- 
vertebrates is  made  up  by  intracellular  free  amino  acids.  Furthermore,  there  are 
many  studies  demonstrating  that  these  free  amino  acids  are  utilized  as  osmotic 
solute  during  salinity  stress  (see  the  reviews  by  Holden,  1 962  for  access  to  the  early 
literature,  more  recently,  Gilles,  1978;  Pierce  and  Amende,  1981). 

The  intracellular  amino  acid  concentration  in  a  euryhaline  invertebrate  adapted 
to  sea  water  can  easily  be  700-800  mA/  (see  for  example  Pierce,  1971;  Costa  et  al., 
1980).  The  amino  acid  pool  size  alone  does  not  always  imply  salinity  tolerance.  For 
example,  the  ascoglossan  opthistobranch  Elysia  chlorotica  which  has  the  widest 
salinity  tolerance  yet  discovered  for  an  osmoconformer  (24-2480  mosm)  has  a  tiny 
amino  acid  pool  (30  /umoles/gm  dry  wt.  in  sea  water)  (Pierce  et  al.,  1982).  Still,  in 
most  cell  types  a  specific  portion  of  the  amino  acid  pool  size  declines  drastically 
with  acclimation  to  a  reduced  salinity.  The  amino  acids  utilized  vary  from  cell  type 
to  cell  type  and  from  species  to  species  but  always  seem  to  be  some  combination 
of  non-essential  amino  acids.  Glycine,  alanine,  proline,  glutamate,  taurine,  occa- 
sionally aspartate  and  glutamine  are  the  usual  amino  acids  involved  (reviewed  by 
Gilles,  1978;  Pierce  and  Amende,  1981). 

Amino  acid  mediated  volume  regulatory  mechanisms  —  older  studies 

Various  aspects  of  amino  acid  mediated  volume  regulation  have  been  studied 
in  a  variety  of  species  and  cell  types.  Of  these,  one  of  the  more  persistent  investi- 
gations into  the  mechanisms  involved  in  the  regulatory  process  has  been  accom- 
plished using  two  molluscan  tissues  as  model  systems:  the  isolated  myocardium  of 
the  ribbed  mussel,  Modiolus  demissus,  and  the  red  blood  cell  of  the  blood  clam, 
Noetia  ponderosa.  The  results  of  these  investigations  have  indicated  that  in  response 
to  low  salinity,  cell  volume  regulation  is  accomplished  by  an  efflux  of  specific  amino 
acids  from  the  cell  (Pierce  and  Greenberg,  1972,  1973,  1976;  Amende  and  Pierce, 
1980).  The  entire  decrease  in  intracellular  amino  acid  concentration  is  accounted 
for  by  the  efflux.  Thus,  there  is  little,  if  any,  intracellular  amino  acid  catabolism  nor 
protein  synthesis  which  occurs  as  part  of  the  volume  regulatory  event.  There  is  some 
evidence  that  the  amino  acids  may  be  catabolized  after  release  from  the  cells.  This 
is  reflected  by  increases  in  both  blood  ammonia  concentrations  and  external  am- 
monia excretion  rates  (for  example  Bartberger  and  Pierce,  1976;  Mangum  et  al., 
1976)  which  follow  the  appearance  of  a  pulse  of  amino  acids  following,  in  turn,  an 
external  salinity  decrease  (Bartberger  and  Pierce,  1976).  There  is  also  some  evidence 
that  the  amino  acids  once  released  from  the  cells  are  sequestered  in  blood  proteins 
for  future  osmotic  uses  (Gilles,  1977;  Boone  and  Schoffeniels,  1979;  Pequeux  et  al., 
1979)  although  this  may  be  a  phenomenon  peculiar  to  the  arthropods. 

The  amino  acid  efflux  is  initiated  by  the  osmotic  pressure  change  rather  than 
the  concomitant  external  ionic  concentration  decrease.  On  the  other  hand,  the  re- 
establishment  of  normal  membrane  permeability  to  amino  acids  (hence,  the  mag- 
nitude and  duration  of  the  efflux)  is  dependent  upon  external  divalent  cation  con- 
centrations, ATP  concentration,  and  temperatures  and  is  independent  of  mono- 
valent  cation  concentrations  (Pierce  and  Greenberg,  1973,  1976;  Watts  and  Pierce, 
1978a;  Amende  and  Pierce,  1980;  Otto  and  Pierce,  1981b).  Thus,  both  an  ionic  and 
metabolic  component  of  the  efflux  control  mechanism  have  been  demonstrated. 
Further,  the  M.  demissus  myocardial  sarcolemma  contains  substantial  divalent  cat- 


410  SIDNEY   K.   PIERCE 

ion  requiring  adenosine  triphosphatase  (ATPase)  activity  (Watts  and  Pierce,  1978b). 
Inhibition  or  potentiation  of  this  ATPase  activity  produced  a  correlative  potentiation 
or  inhibition  respectively  of  the  amino  acid  efflux  from  the  intact  heart  (Watts  and 
Pierce,  1978c).  These  results  led  to  the  hypothesis  that  the  physiological  basis  of  cell 
volume  regulation,  and  thereby  of  low  salinity  tolerance,  in  osmoconforming  marine 
invertebrates  rests  with  a  membrane  bound  divalent  ATPase  which  controls  amino 
acid  permeability  over  a  wide  range  of  external  divalent  ion  concentrations  (Pierce 
and  Greenberg,  1973;  Watts  and  Pierce,  1978c;  Amende  and  Pierce,  1980;  Pierce 
and  Amende,  1981).  Although  no  other  invertebrate  cell  type  had  been  studied  in 
this  detail  up  to  that  point,  these  results  were  generally  confirmed  by  others  (Gilles 
and  Pequeux,  1981;  Pierce  and  Amende,  1981).  At  present  there  are  still  no  data 
establishing  cause  and  effect  between  the  divalent  ATPase  and  amino  acid  efflux 
control,  only  correlations  are  established.  Furthermore  the  mechanism  of  ATPase 
action  is  unknown,  but  most  hypotheses  suggest  a  chemo-mechanical  system  of 
permeability  control  such  as  that  found  by  earlier  studies  of  mammalian  cells  (Wins 
and  Schoffeniels,  1966;  Bowler  and  Duncan,  1967;  Rosenthal  el  al,  1970;  Palek  et 
al,  1971;  Rorive  and  Kleinzeller,  1972;  Quist  and  Roufogalis,  1976). 

As  the  evaluation  of  these  ideas  for  generality  began,  some  important  results 
appeared.  First,  a  comparison  of  the  two  molluscan  cellular  responses  to  hypoos- 
motic  stress  indeed  indicates  similar  characteristics  (ATP  and  divalent  cation  re- 
quirements for  example),  but  also  some  interesting  differences.  The  amino  acid 
efflux  from  the  M.  demissus  myocardial  cells  involves  only  certain  of  the  many 
available  intracellular  amino  acids.  In  these  cells  the  permeability  change  seems  to 
be  quite  specific.  In  contrast,  the  efflux  from  the  N.  ponderosa  blood  cells  is  similar 
in  composition  to  the  intracellular  pool.  The  significance  of  this  difference  between 
the  two  species  is  not  clear  although  it  presents  the  possibility  that  extremely  eu- 
ryhaline  animals  (such  as  M.  demissus)  are  so  as  a  result  of  a  highly  selective  per- 
meability system  allowing  for  both  specific  solute  efflux  and  intracellular  solute 
conservation.  Results  with  cell  types  from  other  euryhaline  animals  (for  example, 
red  coelomocytes  from  the  polychaete  Glycera  dibranchiata  [Costa  et  al,  1980],  M. 
demissus  myocardium  [Pierce  and  Greenberg,  1972],  Rangia  cuneata  myocardium 
[Otto  and  Pierce,  198 la])  indicate  a  selectively  permeable  volume  control  system. 
Second,  and  of  greater  importance,  is  that  recent  studies  clearly  indicate  the  in- 
volvement of  inorganic  ions  in  cell  volume  regulation  by  invertebrate  cell  types.  In 
addition,  in  some  extremely  euryhaline  species  this  ionic  component  plays  a  major 
role  in  the  regulation.  The  majority  of  studies  which  have  produced  these  results 
have  been  done  on  invertebrate  neurons. 

In  spite  of  the  intensity  with  which  nervous  function  has  been  investigated,  there 
are  surprisingly  few  data  on  the  effects  of  osmotic  variation  on  neurons.  The  volume 
regulatory  response  of  axons  to  hypoosmotic  stress  seems  to  be  quite  similar  to  that 
found  in  other  cell  types  (see  above).  For  example,  isolated  Callinectes  axons  swell 
during  exposure  to  a  hypoosmotic  stress  and  then  return  toward  the  initial  volume 
utilizing  a  mechanism  that  requires  Ca2+  and  ATP  (Gerard,  1975).  Axons  from 
other  euryhaline  Crustacea  also  show  similar  patterns  of  volume  changes  (Gilles, 
1973;  Kevers  et  al,  1979a).  Although  electrical  recordings  have  not  accompanied 
the  above  studies,  neuronal  cell  volume  regulation  is  accompanied  by  an  adaptation 
in  the  electrical  properties  of  the  cells.  Usually  a  rapid  hyperpolarization  of  the 
membrane  followed  by  a  slower  depolarization  and  reduction  in  excitability  occurs 
following  a  hypoosmotic  stress  (Maia  axons  [Pichon  and  Treherne,  1976].  Sabella 
giant  axons  [Treherne  and  Pichon,  1978],  Mercierella  axons  [Benson  and  Treherne, 
1978a,  b;  Skaer  et  al,  1978],  Mytilus  cerebro-visceral  connective  [Willmer,  1978], 
Mya  cell  bodies  [Beres  and  Pierce,  1979,  1981]). 


INVERTEBRATE  CELL  VOLUME  CONTROL  411 

Many  of  these  studies  were  conducted  over  short  time  courses.  Recordings  made 
for  longer  intervals  after  the  salinity  decrease  indicated  that  the  depolarization  and 
loss  of  excitability  is  transient,  the  time  course  depending  upon  the  magnitude  of 
the  salinity  change  and  presumably  the  time  course  of  volume  regulation.  For  ex- 
ample, the  spontaneous  burst  frequency,  spike  pattern  within  the  burst,  and  resting 
potential  of  follower  cells  in  the  isolated  Limuhis  cardiac  ganglion  all  returned  to 
control  levels  within  2-3  hours  following  a  salinity  change  from  100%  sea  water 
(SW)  to  50%  SW  (Prior  and  Pierce,  1981).  Similar  results  occurred  with  the  cell 
bodies  of  neurons  in  the  visceral  ganglion  of  the  bivalve  of  Mya  arenaria  (Beres  and 
Pierce,  1981)  and  the  salivary  burster  neuron  in  Limax  (Prior,  1981).  Finally,  all 
of  these  responses  are  due  to  the  osmotic  rather  than  ionic  change  that  accompanies 
the  sea  water  dilution.  None  of  the  electrical  changes  occur  if  only  the  ionic  con- 
centration is  reduced  (osmolality  maintained  with  sucrose)  (Beres  and  Pierce,  1981; 
Prior,  1981;  Prior  and  Pierce,  1981). 

Invertebrate  neurons  —  cell  volume  regulation  mediated  by  inorganic  solutes 

Only  a  few  studies  have  examined  neurons  in  this  connection.  Blue  crab  axons 
(Callinectes  sapidus)  volume  regulate  during  hypoosmotic  stress  using  intracellular 
amino  acids  from  a  substantial  amino  acid  pool  (Gerard,  1975;  Gerard  and  Gilles, 
1972).  Osmotic  adaptation  of  other  neurons  involves  at  least  a  partial  role  of  in- 
organic ions  as  osmotic  solute.  For  example,  the  hypoosmotic  adaptation  ofSabella 
penicillus  axons  includes  a  loss  of  intracellular  K+  (estimated  from  resting  potential 
changes  in  response  to  external  K+  variation)  (Treherne  and  Pichon,  1978).  Treherne 
(1980)  has  proposed  that  the  K+  is  lost  as  osmotic  solute.  Somewhat  similar  ionic 
responses  occur  during  the  adaptation  of  both  Mytilus  cerebro-visceral  connective 
axons  (Willmer,  1978)  and  Mercierella  enigmatica  giant  axons  (Benson  and  Treh- 
erne, 1978b)  to  hypoosmotic  stress.  In  these  two  cases,  however,  the  ionic  changes 
alone  cannot  account  for  the  entire  adaptation  (Treherne,  1980),  and  Mytilus,  at 
least,  has  a  substantial  free  amino  acid  pool  (amino  acids  have  not  been  measured 
in  Mercierella).  Finally,  axons  isolated  from  Carcinus  lose  Na+,  K+,  and  Cl  at  least 
transiently  during  volume  regulation  to  a  hypoosmotic  stress  (Kevers  et  #/.,  1979b). 
Carcinus  axons  also  have  a  substantial  intracellular  amino  acid  pool  (Evans,  1973) 
which  is  apparently  utilized  during  volume  regulation  (Kevers  et  ai,  1979a).  Taken 
together,  these  studies  indicate  that  amino  acid  regulation  is  not  the  entire  story  to 
invertebrate  cell  volume  regulation.  There  are  two  other  invertebrate  cell  types  that 
have  been  studied  in  some  detail  with  respect  to  inorganic  solute  utilization  during 
volume  control:  red  coelomocytes  from  the  blood  worm  Glycera  dibranchiata  and 
the  isolated  myocardium  from  Limulus polyphemus.  Both  cell  types  have  produced 
some  intriguing  results. 

Glycera  red  coelomocytes  —  volume  control  by  amino  acids  and  K+ 

The  isolated,  hemoglobin-containing  coelomocytes  of  Glycera  rapidly  volume 
regulate  in  response  to  a  hypoosmotic  stress  (Costa  et  ai,  1980)  (Fig.  2b).  The  amino 
acid  pool  size  is  large  and  decreases  in  content  as  the  cells  volume  regulate.  Fur- 
thermore, volume  regulation  by  the  isolated  coelomocytes  is  accompanied  by  an 
efflux  of  free  amino  acids  from  the  cells  (Fig.  3)  (Costa  et  al,  1980).  The  volume 
regulatory  process  in  these  cells  requires  the  presence  of  extracellular  divalent  cations 
but  is  not  specific;  either  Ca2+  or  Mg2+  will  suffice.  The  volume  regulatory  process 
appears  to  also  be  dependent  upon  the  metabolic  production  of  ATP  (Costa  and 
Pierce,  1982).  None  of  these  results  is  particularly  surprising  based  on  previous 


412 


SIDNEY   K.   PIERCE 


20 


15- 


O 


5- 


0J 


^4. 

fn 


<J 

£> 

O 


600- 


500- 


tn 
"5         40° 

e 


300- 


200- 


4, 


J         LJ 


996 


592 


mOsm  /  Kg  HO 


10  20 

Time  (min) 


60 


FIGURE  3.  Amino  acid  effluxes  from  Glycera  red  coelomocytes  isolated  from  worms  adapted  to 
996  mosm  and  exposed  to  the  osmotic  concentration  indicated  for  40  min  (From  Costa  et  ai,  1980). 

FIGURE  4.  Intracellular  K+  content  in  Glycera  red  coelomocytes  isolated  from  worms  adapted  to 
996  mosm  and  then  exposed  to  996  or  498  mosm  at  time  0  (From  Costa  and  Pierce,  1982). 


studies,   but  intracellular   K+   content   also  changes  during  volume  control   in 
these  cells. 

A  rapid  decrease  in  intracellular  K+  content  occurred  in  hypoosmotically  stressed 
Glycera  coelomocytes.  Within  10  min  of  exposure  to  dilute  media  intracellular  K+ 
content  declined  by  10%  (Fig.  4).  Further,  incubation  of  the  cells  in  Ca2+-  Mg2+- 
free  media  appears  to  disrupt  cellular  control  of  K4  content  as  well  as  volume 
regulation.  Coelomocytes  incubated  in  Ca2+-  Mg2+-free  media  lose  K+  steadily 
regardless  of  the  external  osmotic  concentration.  The  K+  changes  observed  were 
unaffected  by  ouabain  and  could  be  potentiated  by  incubation  in  2-4  dinitrophenol 
(DNP).  This  last  result  is  of  particular  interest  because  in  Glycera  cells  cellular  amino 
acid  content  was  unaffected  by  DNP  not  only  indicating  that  K+  and  the  amino 
acids  leave  the  cells  by  different  means,  but  also  that  the  two  types  of  solute  are 
responding  to  at  least  some  factors  not  held  in  common.  Finally,  the  use  of  K+  as 
an  osmotic  solute  seems  to  be  only  transitory.  While  intracellular  amino  acid  content 
is  markedly  reduced  in  coelomocytes  taken  from  low  salinity  adapted  worms,  K+ 
content  in  these  coelomocytes  is  not  different  from  that  in  cells  taken  from  high 
salinity  adapted  animals  (Costa  and  Pierce,  1982)  (Table  I). 

Limulus  myocardial  cell  volume  regulation  —  Na+,  Cl~,  and  glycine  betaine 

The  remarkable  euryhalinity  of  Limulus  suggests  an  abundant  amino  acid  pool. 
However,  while  the  intracellular  free  amino  acid  pool  of  Limulus  declines  with 
adaptation  to  low  salinity,  it  is  only  a  small  amino  acid  pool  (total  =  100  ^mole/ 
gm  dry  wt.  in  100%  SW  adapted  crabs)  (Robertson,  1970;  Warren  and  Pierce,  1982). 
Furthermore,  amino  acids  efflux  from  that  pool  in  response  to  a  salinity  decrease, 
but  the  efflux  is  much  too  small  to  account  for  volume  regulation  (Prior  and  Pierce, 
1981).  Instead  Limulus  can  tolerate  a  wide  osmotic  concentration  range  without  a 


INVERTEBRATE  CELL  VOLUME  CONTROL 


413 


TABLE  I 

The  intracellular  K+  content  of  red  coelomocytes  taken  from  Glycera  dibranchiata  acclimated 
to  various  salinities. 


Acclimation  osmotic  concentration 
(mosm/Kg  H2O) 


content 


1000 
750 
500 


417  (±24.8)* 
456  (±22.7) 
413  (±23.7) 


*  nmoles/106  cells  (±S.E.). 

**  Intracellular  K+  content  is  the  same  for  all  treatments,  and  thus  the  use  of  K+  as  osmotic  solute 
in  these  cells  is  only  transient  (from  Costa  and  Pierce,  1982). 


large  amino  acid  pool  because  the  cells  regulate  volume  with  a  mechanism  that 
relies  on  inorganic  ions  and  the  quaternary  ammonium  compound,  glycine  betaine. 

The  role  of  quaternary  ammonium  compounds  as  osmotic  solute  was  occa- 
sionally pointed  out  in  the  older  literature  (for  example,  Bricteux-Gregoire  et  al., 
1964).  More  recent  investigators  have  tended  to  ignore  these  potentially  important 
compounds  largely  because  their  identification  and  quantification  was  difficult  and 
rather  imprecise.  Recently  a  high  performance  liquid  chromatographic  analysis  has 
been  developed  which  solves  these  analytical  problems  (Warren  and  Pierce,  1982). 
The  major  quaternary  ammonium  compounds  in  Limulus  cardiac  tissue  are  glycine 
betaine  and  homarine.  Of  these  glycine  betaine  is  quite  high  in  concentration  in 
tissue  taken  from  Limulus  adapted  to  full  strength  sea  water  and  declines  in  con- 
centration in  Limulus  adapted  to  lower  salinities  (Fig.  5)  (Warren  and  Pierce,  1982). 

The  isolated  Limulus  heart  volume  regulates  in  response  to  a  hypoosmotic  stress. 
The  tissue  shows  a  pattern  of  incomplete  volume  recovery  quite  typical  of  the  pattern 
exhibited  by  most  cell  types  (Fig.  6).  However,  no  betaine  appeared  in  the  media 
surrounding  the  volume  regulating  hearts  and,  indeed  the  betaine  content  in  the 
tissue  was  unchanged  (Table  II)  (Warren  and  Pierce,  1982).  Volume  regulation  by 
the  isolated  heart  was  accomplished  without  utilizing  this  major  osmotic  solute. 


600 


500 


-       400 


300 


200 


100 


•  Glycine  betaine 

•  Homarine 


200 


400  6OO 

Salinity  (mosm) 


800 


1000 


FIGURE  5.  Concentrations  of  the  quaternary  ammonium  compounds  glycine  betaine  and  homarine 
in  cardiac  tissue  of  Limulus  adapted  for  at  least  two  weeks  to  the  salinities  indicated  (From  Warren  and 
Pierce,  1982). 


414 


SIDNEY   K.   PIERCE 


140 


940— 940mosm 
940— 400mosm 


8  10 

Hours 


12 


24 


FIGURE  6.  Volume  regulation  by  isolated  Limulus  hearts.  The  hearts  were  removed  from  crabs 
adapted  to  940  mosm  and  then  exposed  directly  to  either  940  or  400  mosm  at  time  0.  The  hearts  were 
weighed  at  the  time  points  indicated  (From  Warren  and  Pierce,  1982). 


Similarly,  K+  concentration  of  the  isolated  cardiac  tissue  changed  only  as  predicted 
by  hydration  changes  (Table  III).  On  the  other  hand,  cellular  Na+  and  Cl  levels 
decreased  far  more  than  cell  hydration  changes  could  account  for  during  the  hy- 
poosmotic  stress  (Table  IV  and  V)  (Warren  and  Pierce,  1982).  Furthermore,  these 
ionic  changes  are  ouabain  independent.  These  results  clearly  show  that  Na+  and  Cl~ 
are  utilized  to  regulate  volume  early  on  by  the  Limulus  cells  and  glycine  betaine 
much  later.  Indeed,  evidence  from  whole  animal  experiments  indicates  that  Na+ 
and  Cl  partially  return  toward  initial  concentrations  as  glycine  betaine  declines  in 
the  Limulus  heart  cells  (Warren  and  Pierce,  1982). 

Volume  regulation  may  result  from  coordination  of  permeability  control  systems 
—  conclusions 

The  results  of  the  Glycera  and  Limulus  studies  taken  together  indicate  that  two 
quite  distinct  solute  permeability  control  mechanisms  are  utilized  by  these  cells 
during  volume  regulation.  The  ions  involved  (Na+,  K4,  or  Cl  depending  upon  the 
cell  type)  respond  to  the  decrease  in  external  ionic  concentrations  which  accompany 
the  salinity  decrease.  The  amino  acid  efflux  is  triggered  by  the  osmotic  change.  The 
ionic  movements  are  not  affected  by  ouabain  indicating  that  the  Na+  pump  is  not 


TABLE  II 

Glycine  betaine  concentrations  in  hearts  isolated  from  Limulus  adapted  to  940  mosm 
and  exposed  to  400  mosm. 


940  mosm 


400  mosm 


6  h 

12  h 
24  h 


599  ±  24* 
621  ±  16 
585  ±  21 


633  ±  15 
631  ±27 
620  ±  21 


*  mmoles/g  dry  wt  ±  S.E. 

The  low  salinity  values  are  not  significantly  different  from  the  high  salinity  controls  (from  Warren 
and  Pierce,  1982). 


INVERTEBRATE  CELL  VOLUME  CONTROL 


415 


TABLE  III 
Intracellular  K+  in  hearts  isolated  from  Limulus  adapted  to  940  mosrn  and  exposed  to  400  mosm. 


mmoles/kg  H2O 


mmoles/kg  dry  wt 


Salinity 


940  mosm 


400  mosm 


Predicted" 


940  mosm 


400  mosm 


6  h 
12  h 

24  h 

112.6  ±  7.5 
113.0  ±  5.7 
114.5  ±  5.3 

74.7  ±  2.9 
83.2  ±  3.2 
88.6  ±  3.5 

75.0  ±  5.2 
73.6  ±  4.3 
79.3  ±  4.6 

458  ±  29 
432  ±  28 
458  ±  20 

432  ±  14 
453  ±  11 
511  ±  13 

*  Calculated  according  to  Freel  el  al.,  1973. 

The  data  are  presented  in  two  ways.  First,  as  concentration  (mmoles/kg  H2O).  A  concentration 
decrease  of  K+  does  occur,  but  only  as  much  as  is  predicted  by  changes  in  tissue  hydration.  Second,  as 
content  (mmoles/kg  dry  wt).  There  is  no  significant  change  in  K+  content  indicating  that  K+  is  not  used 
as  osmotic  solute  (from  Warren  and  Pierce,  1982). 


involved  in  the  process.  It  is  clear  from  the  data  cited  above  that  the  two  types  of 
permeability  systems  can  be  made  to  operate  independently  of  one  another  and  that 
they  often  function  with  very  different  time  courses  in  the  cell.  Nonetheless,  it  is 
also  clear  that  both  solute  control  systems  operate  in  concert  to  control  cell  volume. 
The  mechanism  underlying  this  remarkable  coordination  is  unknown  at  present, 
but  may  be  Ca2+  related.  The  amino  acid  efflux  control  mechanism  requires  Ca2+ 
(see  above),  and  normal  K+  permeability  in  the  Glycera  cells  is  lost  if  Ca2+  is  removed 
(Costa  and  Pierce,  1982).  However,  at  present  little  else  is  known  about  the  char- 
acteristics of  the  ionic  regulatory  systems. 

Finally,  some  comparisons  to  volume  regulatory  systems  found  in  vertebrate 
cells  may  be  instructive.  There  is  no  doubt  that  a  substantial  inorganic  ionic  com- 
ponent is  responsible  for  volume  regulation  in  vertebrate  cells.  Usually  Na+  or  K+ 
or  both  are  utilized  in  a  ouabain  insensitive  volume  regulatory  process  that  occurs 
following  an  osmotic  alteration  (as  opposed  to  steady  state  osmotic  balance  which 
is  usually  ouabain  sensitive)  (reviewed  by  Rorive  and  Gilles,  1979).  Occasionally 
other  ions  are  involved.  For  example,  Necturus  gall  bladder  epithelial  cells  require 
bicarbonate  for  volume  regulation  (Fisher  et  al.,  1981).  In  addition,  although  there 
are  not  yet  a  lot  of  data,  it  seems  that  vertebrate  cells  also  have  an  organic  solute 
component  to  the  cell  volume  regulatory  mechanism.  This  component  utilizes 
amino  acids  or  quaternary  ammonium  compounds  and  is  particularly  obvious  in 


TABLE  IV 
Intracellular  Na+  in  hearts  isolated  from  Limulus  adapted  to  940  mosm  and  exposed  to  400  mosm. 


mmoles/kg  H2O 


mmoles/kg  dry  wt 


Salinity 


940  mosm 


400  mosm 


Predicted* 


940  mosm 


400  mosm 


6  h 
12  h 

237 
228 

.7  ± 
.9  ± 

13.0 
17.6 

79.0  ± 
46.3  ± 

16.9 
6.3 

153.1  ± 
144.5  ± 

8.9 
10.6 

913 
905 

±  51 

±  72 

437 
273 

±  89 
±  34 

*  Calculated  according  to  Freel  et  al.,  1973. 

These  data  are  also  presented  two  ways  (see  Table  III).  There  is  a  substantial  change  in  Na+  con- 
centration which  is  greater  than  that  which  can  be  accounted  for  by  hydration  changes.  This  is  verified 
by  the  Na+  content  data  which  also  shows  very  significant  decreases  during  hypoosmotic  stress.  Therefore, 
Na+  is  used  as  osmotic  solute  (from  Warren  and  Pierce,  1982). 


416  SIDNEY   K.   PIERCE 

TABLE  V 
Intracellular  CT  in  hearts  isolated  from  Limulus  adapted  to  940  mosm  and  exposed  to  400  mosm. 

mmoles/kg  H2O  mmoles/kg  dry  wt 


Salinity  940  mosm  400  mosm  Predicted*  940  mosm  400  mosm 


6  h 

199 

9  ± 

16.0 

60.3 

±  8.7 

124.7  ± 

9.4 

780 

±  52 

352 

±  56 

12  h 

195 

,8  ± 

13.5 

39.5 

±  4.6 

135.8  ± 

12.8 

762 

±  48 

221 

±  26 

24  h 

201 

,6  ± 

15.3 

38.8 

±  5.1 

141.0  ± 

8.3 

834 

±  76 

213 

±  24 

*  Calculated  according  to  Free!  el  ai,  1973. 

Cl~  concentration  decreased  much  more  than  hydration  changes  could  account  for  and  Cl  content 
also  showed  large,  significant  decreases.  Therefore  Cr,  like  Na+  (Table  IV),  is  regulated  in  the  heart  cells 
in  response  to  hypoosmotic  stress  (from  Warren  and  Pierce,  1982). 


vertebrate  species  that  spend  all  or  part  of  their  lives  in  water  (for  example  marine 
toad  [Bufo  viridis]  skeletal  muscle  [Gordon,  1965],  flounder  [Pleuronectes  flesus} 
red  cells  [Fugelli,  1967],  Myxine  muscle  cells  [Cholette  and  Gagnon,  1973],  skate 
[Raja  erinacea]  and  stingray  [Dasyatis  sabina]  tissues  [Boyd  et  ai,  1977],  skate 
[Raja  erinacea]  erythrocytes  and  muscle  [Goldstein.  1981]).  Other  studies  have 
demonstrated  utilization  of  organic  osmotic  solute,  primarily  taurine,  in  higher  ter- 
restrial vertebrates  including  humans.  For  example,  intracellular  taurine  concentra- 
tions respond  to  the  plasma  osmolality  changes  that  occur  during  hypo-  or  hyper- 
natremia  in  both  mammalian  brain  and  heart  cells  (Thurston  et  al.,  1980,  1981; 
also  reviewed  by  Pollock  and  Arieff,  1980).  Ehrlich  ascites  cells  also  utilize  taurine 
for  volume  control  (Hendil  and  Hoffman,  1974;  Hoffman,  1978).  At  present  little 
is  known  about  the  mechanisms  utilized  to  control  the  organic  solutes  in  these  cell 
types.  Nonetheless,  the  historic  intracellular  osmotic  solute  differences  held  to  occur 
between  vertebrates  and  invertebrates  may  be  a  strawman.  There  is  a  growing  body 
of  information  indicating  that  both  types  of  solute  are  utilized  by  all  cells,  and 
differences  are  in  magnitude  rather  than  kind.  If  this  turns  out  to  be  true,  then  the 
cells  of  euryhaline  invertebrates  may  become  important  as  well  as  interesting  models 
of  osmotic  function  as  a  consequence  of  their  remakable  abilities  of  cell  hydration 
control. 

ACKNOWLEDGMENTS 

Some  of  the  studies  reported  here  were  supported  by  N.I.H.  Grant  #  GM-2373 1 . 
This  paper  is  Contribution  No.  1 90  from  the  Tallahassee,  Sopchoppy  &  Gulf  Coast 
Marine  Biological  Association,  Inc. 

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EFFECTS  OF  ENZYMATIC  AND  NONENZYMATIC  PROTEINS  ON 

ARBACIA   SPERMATOZOA:   REACTIVATION  OF  AGED  SPERM   AND 

THE  INDUCTION  OF  POLYSPERMY* 

PHILIP  DUNHAM.   LEONARD  NELSON,   LESLIE   VOSSHALL,  AND 

GERALD  WEISSMANN 

Marine  Biological  Laboratory,  Woods  Hole.  MA  02543 

ABSTRACT 

Arbacia  sperm  become  inactive  after  dilution  in  sea  water.  We  have  shown  that 
any  of  six  proteins  reactivated  the  aged  sperm  as  judged  by  their  fertilizing  capacity 
or  their  motility.  In  suspensions  of  inactive  sperm  in  which  the  mean  fertilizing 
capacity  was  less  than  3%,  brief  incubation  with  any  of  the  proteins  at  0.5  mg/ml 
stimulated  fertilizing  capacity  to  70-90%.  Reactivation  by  the  proteins  was  detected 
at  concentrations  lower  than  2  Mg/ml.  All  six  of  the  proteins  also  stimulated  motility 
of  aged  sperm  by  30-70%. 

The  normal  block  to  polyspermy  may  involve  inactivation  of  sperm  by  sub- 
stances released  from  the  eggs  during  the  fertilization  reaction.  All  six  proteins  tested 
on  inactive  sperm  were  also  shown  to  induce  polyspermy  in  mixtures  of  eggs  and 
fresh  sperm.  Whereas  in  control  mixtures  with  polyspermic  cleavage  of  ~1%  of 
eggs,  proteins  at  2  mg/ml  induced  5-50%  polyspermic  cleavage,  and  induction  of 
polyspermy  was  detected  at  5  Mg/ml. 

The  six  proteins  showing  activity  included  enzymes  and  also  the  relatively  inert 
gelatin.  The  concentration  dependence  was  upon  weight/volume  and  not  molarity. 
Though  the  mode  of  action  is  unclear,  it  must  be  rather  nonspecific,  and  is  certainly 
not  dependent  on  enzymatic  activity.  The  same  mode  of  action  is  likely  for  activation 
of  aged  sperm  and  induction  of  polyspermy. 


INTRODUCTION 

Sea  urchin  sperm  suspended  in  sea  water  for  a  few  hours  become  immotile  and 
lose  their  ability  to  fertilize  eggs  (Gemmill,  1900;  Gray,  1928a;  Rothschild  and  Tyler, 
1954;  Bishop,  1962;  Branham,  1966;  Mann,  1964;  Nelson,  1967).  Sir  James  Gray 
(1928a)  entertained  the  possibility  that  this  "senescence"  might  be  reversible.  Al- 
though inactivation  was  ascribed  to  a  "loss  of  energy  reserves"  (Gemmill,  1900; 
Tyler,  1953),  it  has  been  suggested  that  inactive,  senescent  Arbacia  sperm  can  be 
reactivated  by  treatments  which  would  not  be  expected  to  replenish  "energy  re- 
serves" (e.g.  dilution  by  fresh  sea  water  [Gray,  1928a]  or  suspension  in  sea  water 
in  which  eggs  had  been  incubated  [Cohn,  1918;  Hathaway,  1963]).  However,  no 
satisfactory  explanations  have  been  offered  which  explain  the  well-documented  in- 
activation, or  the  less  clearly  denned  reactivation  of  sperm.  Thus,  for  example,  an 
inhibitory  effect  of  heavy  metals  has  been  invoked  to  explain  the  inactivation  of 
sperm  (Rothschild  and  Tyler,  1954).  While  metals  certainly  may  inhibit  motility, 

Received  3  December  1981;  accepted  7  September  1982. 

*  \\'e  dedicate  this  paper  to  the  late  H.  Burr  Steinbach,  the  mentor  of  two  of  us  (PD  &  LN). 

Abbreviations:  see  Table  I. 

420 


PROTEIN  ACTIVATION  OF  ARBACIA  SPERM  421 

their  removal  is  unlikely  to  be  the  basis  for  the  increase  in  oxygen  consumption 
upon  dilution  in  fresh  sea  water,  the  "dilution  effect"  (Gray,  1928b).  Possible  clues 
come  from  studies  in  which  a  wide  variety  of  agents  have  been  shown  either  to 
increase  the  motility  of  freshly  diluted  Arbacia  sperm  or  slow  the  onset  of  inacti- 
vation  (?.#.,  Branham,  1966;  Steinbach,  1966;  Tyler  and  Tyler,  1966;  Nelson,  1972a, 
1978;  see  also  Steinbach  and  Dunham,  1961).  Similar  observations  on  motility  have 
been  made  on  sperm  from  various  mammalian  and  avian  species  (Schindler  and 
Nevo,  1962;  Wales  and  White,  1962;  Liess  and  Grove,  1963;  VanDemark  and 
Koyama,  1963;  Garbers  et  al,  1971;  Bavister,  1981). 

The  normal  inhibition  of  polyspermy  in  Arbacia  eggs  and  those  of  many  other 
species  has  long  been  recognized  but  remains  poorly  understood.  Two  general  types 
of  mechanisms  might  be  involved:  1)  the  surface  of  the  egg,  or  some  portion  of  it, 
may  be  altered  subsequent  to  binding  or  fusion  of  one  sperm,  thereby  reducing  the 
probability  of  penetration  of  additional  sperm;  2)  subsequent  to  contact  with  one 
sperm  the  egg  may  release  an  agent  or  agents  which  reduce  the  fertilizing  capacity 
of  neighboring  sperm.  Mechanisms  of  both  types  have  been  proposed.  For  example, 
F.  R.  Lillie  described  the  reversible  agglutination  of  Arbacia  sperm  by  a  substance 
released  from  eggs  which  he  called  fertilizin  (Lillie,  1913,  1919).  Lillie  also  appre- 
ciated that  the  egg's  cortical  reaction  is  too  slow  to  be  the  only  process  at  the  surface 
of  the  egg  operating  to  prevent  polyspermy  (see  recent  reviews  containing  discussions 
of  polyspermy  by  Austin,  1978;  Epel,  1978;  Schuel,  1978;  and  Dale  and 
Monroy,  1981). 

Doubts  have  been  expressed  about  the  existence  of  a  rapid  block  to  polyspermy 
in  sea  urchin  eggs  (Hagstrom  and  Allen,  1956;  Dale  and  Monroy,  1981).  A  block 
to  polyspermy  associated  with  electrical  depolarization,  first  proposed  by  Gray  in 
1922,  has  recently  been  demonstrated  in  Strongylocentrotus  eggs  (Jaffe,  1976);  the 
depolarization  and  associated  block  to  polyspermy  have  been  suggested  to  depend 
on  Na  (Schuel  and  Schuel,  1981).  However,  the  validity  of  these  conclusions  has 
been  called  into  question  (Dale  and  Monroy,  1981). 

It  was  shown  nearly  a  century  ago,  and  confirmed  many  times  since,  that  a  wide 
variety  of  chemical  agents  can  induce  polyspermy  in  sea  urchin  eggs  (Hertwig  and 
Hertwig,  1887;  Just,  1928;  Clark,  1936;  Rothschild,  1954;  Hagstrom,  1956;  Schuel 
et  al.,  1976;  Coburn  et  al.,  1981).  Polyspermy  has  now  been  observed  in  a  wide 
range  of  animals  (mammals  as  well  as  invertebrates).  The  nature  of  agents  with  such 
reactivity  is  so  diverse  as  to  support  no  single  proposed  mechanism  for  polyspermy; 
rather,  the  diversity  suggests  multiple  mechanisms  by  which  polyspermy  is  induced 
and  therefore  redundant  mechanisms  for  the  normal  block  to  polyspermy.  For 
example,  Hertwig  and  Hertwig  (1887)  and  Hagstrom  and  Allen  (1956)  induced 
polyspermy  with  nicotine  and  Clark  ( 1 936)  and  Hagstrom  ( 1 956)  did  so  with  strych- 
nine; Nelson  demonstrated  stimulation  of  motility  of  freshly  diluted  sperm  by  na- 
nomolar  concentrations  of  nicotine  (Nelson,  1978)  and  by  micromolar  concentra- 
tions of  strychnine  (Nelson,  1972a).  However,  the  higher  concentrations  which  may 
have  been  necessary  for  induction  of  polyspermy  (e.g.  Clark,  1936)  inhibit  motility 
(Nelson,  1972a,  1978;  JafFe,  1980). 

An  agent  with  very  different  reactivity,  trypsin  inhibitor  from  soy  beans,  has 
been  shown  to  induce  polyspermy  (Hagstrom,  1956;  Vacquier  et  al.,  1972;  Schuel 
et  al.,  1976).  This  agent  might  act  by  interfering  with  the  cortical  reaction  (one  of 
the  initial  events  in  fertilization),  consistent  with  a  role  of  an  esteroprotease  in  this 
reaction  (Grossman  et  al.,  1973).  In  another  intriguing  observation,  polspermy  was 
induced  by  catalase  (Coburn  et  al.,  1981),  suggesting  that  the  block  to  polyspermy 
is  due  to  release  of  H2O2  from  the  eggs  during  the  fertilization  reaction. 


422  P.   DUNHAM   ET  AL. 

We  have  found  that  any  of  several  proteins  can  reactivate  inactive  sperm.  Reac- 
tivation was  judged  from  measurements  of  fertilizing  capacity  and  of  motility.  The 
same  proteins  also  induced  polyspermy  over  a  similar  range  of  concentrations.  The 
diverse  properties  of  the  proteins  (from  the  enzyme  catalase  to  the  relatively  inert 
gelatin)  suggest  that  their  mode  of  action  is  nonspecific.  Our  results  represent  the 
first  clear  demonstration  of  reactivation  of  the  fertilizing  capacity  of  inactive  sperm. 
We  also  provide  evidence  that  induction  of  polyspermy  and  reactivation  of  sperm 
have  a  similar  basis.  Hov/ever,  it  is  probable  that  more  than  one  mechanism  exists 
for  induction  of  polyspermy  (and  therefore  that  there  is  more  than  one  mechanism 
for  the  physiological  block  to  polyspermy).  Finally,  the  nature  of  our  effective  agents 
requires  a  reexamination  of  mechanisms  which  have  previously  been  proposed  for 
the  modulation  of  the  activity  of  sperm. 

MATERIALS  AND  METHODS 

Gametes.  Spermatozoa  and  eggs  were  obtained  from  mature  sea  urchins  (Arbacia 
punctulatd)  collected  by  the  Department  of  Marine  Resources  of  the  Marine  Bio- 
logical Laboratory. 

Sperm:  Electrodes  from  a  12  v  A.C.  source  were  placed  across  the  aboral  surface 
of  a  male  sea  urchin  for  30  sec  or  less.  The  sperm  released  were  rinsed  into  sea  water 
(~  15  ml).  Numbers  of  sperm  per  ml  were  determined  by  absorbance  of  light  at  540 
nm  in  a  Spectronic  20  Colorimeter  (Bausch  and  Lomb)  (Nelson,  1972a). 

Eggs:  Female  sea  urchins  were  inverted  over  beakers  of  sea  water  (50  ml)  and 
injected  periviscerally  with  ~1  ml  of  0.5  M  KC1.  The  eggs  released  were  washed 
twice  in  sea  water  by  suspension  and  sedimentation  at  1  X  g.  Numbers  of  eggs/ml 
were  calculated  from  the  packed  volume  of  eggs  after  centrifugation  to  constant 
volume  with  a  hand  centrifuge  and  the  mean  diameter  of  Arbacia  eggs  (~75  /urn; 
Harvey,  1956). 

Inactivation  of  sperm  by  aging.  Suspensions  of  sperm  diluted  in  sea  water  to 
about  30  X  106  sperm/ml  were  allowed  to  stand  for  one  to  two  days  at  room  tem- 
perature (22-25°C). 

Fertilizing  capacity  of  sperm.  As  a  measure  of  the  function  of  sperm,  fresh  and 
inactivated,  their  capacity  to  fertilize  eggs  was  measured.  The  method  was  similar 
to  that  of  Lillie  (1915).  These  assays  were  carried  out  in  plastic  Petri  dishes  (35  mm 
X  10  mm)  at  room  temperature  in  a  total,  final  volume  of  2  ml.  Appropriate  volumes 
of  sperm  suspension  (0.05-0.2  ml)  were  added  to  give  ~  106  sperm/ml,  final  density. 
Agents  to  be  tested  for  their  effect  on  fertilizing  capacity  were  then  added,  and  the 
mixtures  were  incubated  at  room  temperature,  usually  for  6  minutes.  Then  eggs 
were  added  (0.1-0.2  ml  of  stock  suspension)  to  a  final  density  of  25,000  eggs/ml. 
After  incubation  for  5  minutes,  fertilizing  capacity  of  the  sperm  was  assayed  by 
counting  the  number  of  eggs  (in  a  field  of  100)  with  a  raised  fertilization  membrane. 
(In  some  experiments  the  eggs  were  counted  again  after  90  min  for  2-cell  stages  as 
a  measure  of  "normal"  fertilization.)  Bright  field  illumination  in  a  compound  mi- 
croscope was  used  at  low  power.  During  this  study,  49,400  eggs  in  all  were  scored 
(c.f.  Weissmann,  1981). 

We  observed  that  SBTI  modified  the  cortical  reaction  which  occurs  upon  fer- 
tilization, confirming  the  observations  of  others  (see  Epel,  1978,  and  Schuel,  1978). 
The  lifting  of  the  fertilization  membrane  was  much  less  pronounced  than  in  control 
eggs.  However  fertilization  was  not  prevented  by  SBTI  and  subsequent  divisions 
were  not  modified.  None  of  the  other  proteins  tested  modified  the  cortical  reaction. 


PROTEIN  ACTIVATION  OF  ARBAC1A  SPERM  423 

Motility  of  sperm.  This  was  determined  by  a  method  described  earlier  (Nelson, 
1972b).  Aged  sperm  were  first  incubated  (~6  min)  with  agents  to  be  tested  for  their 
effect  on  motility.  Then  the  sperm  suspension  (at  4-8  X  106/ml)  was  placed  in  a 
low  centrifugal  field  ( 1 20  X  g)  at  room  temperature  for  4  minutes.  Under  these 
conditions  (in  which  formaldehyde-killed  sperm  do  not  sediment),  motile  sperm 
tend  to  move  in  a  centrifugal  direction  and  the  immotile  sperm  remain  in  the 
supernatant  suspension.  Thus  the  optical  density  (at  540  nm  in  a  Spectronic  20 
Colorimeter)  of  the  supernatant  suspension  (containing  the  immotile  cells)  is  in- 
versely related  to  motility  (Nelson,  1972b). 

Polyspermy.  Polyspermy  was  assayed  in  plastic  Petri  dishes  set  up  as  described 
above  for  measurement  of  fertilizing  capacity.  Sperm  were  incubated  (6  min)  with 
agents  to  be  tested  for  their  promotion  of  polyspermy.  Then  eggs  were  added  and 
the  mixtures  were  incubated  for  45-60  minutes.  In  all  cases  at  least  90%  of  the  eggs 
were  fertilized,  and  at  least  55%  (and  generally  more  than  80%)  of  the  eggs  cleaved, 
either  reaching  the  normal  two-cell  stage,  or  being  readily  recognizable  as  an  aberrant 
form  typical  of  polyspermy  (Just,  1928;  Clark,  1936).  Scoring  was  made  of  fields 
of  100  eggs  for:  a)  unfertilized  eggs;  b)  fertilized  eggs,  1-cell  stage;  c)  normally  fer- 
tilized eggs.  2-cell  stage;  and  d)  polyspermic  eggs. 

Proteins.  The  proteins  tested  for  their  effects  on  spermatozoan  function  were 
added  to  the  assay  suspensions  from  stock  solutions  made  in  sea  water  (up  to  10 
mg/ml).  Table  I  lists  the  proteins  employed,  their  approximate  molecular  weights, 
and  their  commercial  sources. 

Statistical  tests.  The  randomization  test  for  matched  pairs  (two  tailed)  was  used 
to  determine  levels  of  significance  of  difference  (P)  from  controls  caused  by  treat- 
ments with  proteins.  This  is  a  nonparametric  test  with  100%  power  efficiency  (Siegel, 
1956).  Standard  errors  of  means  (SEMs),  not  used  in  tests  for  significance  of  dif- 
ferences, are  shown  to  indicate  variability  between  experiments.  The  number  of 
separate  experiments  (on  different  preparations  of  cells)  is  given  by  "n". 

RESULTS 

Reactivation  by  proteins  of  aged  sperm:  fertilizing  capacity.  We  confirmed  that 
Arbacia  sperm  diluted  in  sea  water  and  aged  for  a  number  of  hours  become  inactive 

TABLE  I 

Proteins  employed  in  studies  on  function  of  Arbacia  spermatozoa,  the  approximate  molecular  weights 
of  the  proteins,  and  their  commercial  sources. 


Protein 

Molecular  weight 

Commercial  source 

Abbreviation 

catalase  (prepared  from 

250,000 

Sigma  Chemical  Co., 

CAT 

bovine  liver) 

St.Louis,  MO 

crystalline  bovine  serum 

60,000 

Sigma 

BSA 

albumin 

Cohn  fraction  V  (from 

60,000 

Sigma 

CFV 

bovine  serum) 

superoxide  dismutase 

32,000 

Miles  Laboratories  Ltd., 

SOD 

Rep.  of  S.A. 

Soy  bean  trypsin  inhibitor 

21,000 

Sigma 

SBTI 

(type  I-S) 

gelatin  (granular) 

(indeterminate) 

Matheson  Coleman  & 

GEL 

Bell,  Norwood,  OH 


424 


P.   DUNHAM   ET  AL. 


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FIGURE  1.  Reactivation  of  the  fertilizing  capacity  of  aged  Arbacia  sperm  by  various  proteins,  all 
at  0.5  mg/ml.  Procedures  for  inactivation  of  sperm  by  aging  and  for  determination  of  fertilizing  capacity 
are  given  in  Materials  and  Methods.  Error  bars  indicate  SEMs;  n,  numbers  of  experiments.  P  is  the  level 
of  significance  of  difference  from  aged  sperm  not  treated  with  protein  (randomization  test  for  matched 
pairs). 


as  judged  by  their  fertilizing  capacity.  We  then  discovered  that  brief  incubation  of 
these  aged  sperm  with  any  of  several  proteins  dramatically  restored  their  fertilizing 
capacity.  Figure  1  shows  measurements  of  fertilizing  capacity  of  fresh  sperm,  aged 
sperm  (one  or  two  days),  and  aged  sperm  incubated  6  minutes  with  six  different 
proteins  (all  at  0.5  mg/ml),  both  enzymatic  (CAT  and  SOD)  and  nonenzymatic.  In 
preliminary  experiments,  one  other  protein,  ovalbumin,  also  reactivated  aged  sperm. 
No  other  proteins  were  tested. 

Preliminary  determinations  of  the  time  course  of  reactivation  indicated  that  the 
full  effect  was  achieved  well  before  6  minutes.  Unfortunately  the  time  required  for 
fertilization  by  fully  active  sperm  makes  an  accurate  determination  of  the  time 
course  of  reactivation  impossible. 

Figure  2  shows  the  effect  on  fertilizing  capacity  of  aged  sperm  of  the  proteins 
in  Figure  1  as  a  function  of  protein  concentration  (weight/volume).  Reactivating 
activity  was  detectable  at  5  Mg/ml  or  less.  The  curves  for  the  various  proteins  are 
similar  with  concentrations  expressed  as  weight/volume  despite  the  wide  range  of 
their  molecular  weights  (21,000-250,000;  see  Table  I). 

Reactivation  of  aged  sperm:  motility.  Figure  3  shows  measurements  of  motility 
of  aged  sperm  reactivated  by  brief  incubation  with  each  of  the  six  proteins  used  to 
reactivate  fertilizing  capacity.  The  motility  of  the  aged  sperm  was  about  25%  of  the 
motility  of  freshly  diluted  sperm.  All  of  the  proteins  increased  the  motility  of  aged 
sperm.  Reactivation,  judged  by  motility,  is  less  dramatic  in  quantity  than  the  reac- 
tivation of  fertilizing  capacity,  but  it  is  striking  nevertheless. 

Despite  reactivation  of  aged,  inactive  sperm  by  proteins,  we  observed,  in  a  pre- 
liminary experiment,  that  aging  the  sperm  in  the  presence  of  the  proteins  did  not 
protect  them  from  eventual  inactivation  as  judged  by  their  motility. 

Induction  of  polyspermy  by  proteins.  Table  II  shows  the  results  of  three  typical 
experiments  on  induction  of  polyspermy  by  three  proteins.  In  addition  to  the  results 
on  polyspermy,  Table  II  shows  that  the  treatment  with  proteins  did  not  affect  fer- 
tilization or  cleavage.  That  the  variability  in  per  cent  polyspermy  among  these  three 


PROTEIN  ACTIVATION  OF  ARBACIA  SPERM 


425 


80  r- 


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20 


Protein 

_n_ 

• 

—  cotolose 

2 

D 

—  cryst  BSA 

2 

A 

—  Cohn  froct  ¥ 

2 

O 

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2 

A 

-  SBTI 

1 

• 

—  gelatin 

1 

1.7 


5  17  50 

Protein  concentration:  ^.o/ml 


167 


500 


FIGURE  2.  Reactivation  by  proteins  of  fertilizing  capacity  of  aged  Arbacia  sperm  as  a  function  of 
protein  concentration  (^g/ml).  Procedures  for  inactivation  and  for  determination  of  fertilizing  capacity 
are  given  in  Materials  and  Methods.  Protein  concentrations  on  the  abscissa  are  plotted  in  a  logarithmic 
scale.  The  inset  shows  the  symbols  for  the  proteins  and  n,  the  numbers  of  experiments  for  each. 


experiments  was  great  is  indicated  by  the  standard  errors.  In  these  three  experiments, 
however,  in  no  instance  was  the  level  of  polyspermy  induced  by  a  protein  in  a 
suspension  of  sperm  and  eggs  less  than  2-fold  greater  than  its  control. 

Figure  4  shows  levels  of  polyspermy  induced  by  the  six  proteins  (all  at  2  mg/ml) 
in  a  series  of  experiments  (not  every  protein  was  tested  in  each  experiment).  Again, 


200 


o 


9) 

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150 


100 


CD 


CD 

cn 


o 
o 


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o> 


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o 


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FIGURE  3.  Motility  of  aged  Arbacia  sperm  reactivated  with  proteins,  presented  relative  to  the 
motility  of  aged  sperm,  and  determined  as  described  in  Materials  and  Methods.  The  motility  of  aged 
sperm  not  treated  with  proteins,  set  at  100,  was  approximately  25%  of  the  motility  of  fresh  sperm. 
Numbers  of  experiments  are  shown;  the  error  bars  show  SEMs  for  BSA,  SBTI,  and  CAT  (n  >  3),  and 
total  ranges  for  SOD  and  CFV  (n  =  2). 


426 


P.   DUNHAM   ET  AL. 


TABLE  II 
Induction  ofpolyspermy  by  proteins  in  mixtures  of  Arbacia  sperm  and  eggs. 


Condition 

Per  cent 
fertilized  eggs 

Per  cent  cleavage 
among  fertilized  eggs 

Per  cent  polyspermy 
among  cleaved  eggs 

control 
catalase 
gelatin 
crystalline  albumin 

99.0  ±  0.6 
98.3  ±  1.7 
98.0  ±  2.0 
99.3  ±  0.3 

93.7  ±  3.8 
92.3  ±  2.3 
93.3  ±  1.8 
93.3  ±  2.0 

2.3  ±  1.4 
22.3  ±  9.4 
9.3  ±  2.0 
17.1  ±  5.9 

Shown  are  the  per  cent  of  eggs  fertilized,  the  per  cent  of  fertilized  eggs  which  had  undergone  cleavage 
(either  to  the  normal  2-cell  stage  or  to  aberrant  polyspermic  forms),  and  the  per  cent  of  cleaved  eggs 
which  were  polyspermic.  Results  are  from  suspensions  of  sperm  and  eggs  incubated  with  catalase,  gelatin, 
or  crystalline  albumin  (all  at  2  mg/ml),  and  control  suspensions.  Values  are  means  ±  SEMs  from  3 
experiments. 


in  no  instance  was  the  per  cent  polyspermy  induced  by  a  particular  protein  less  than 
2-fold  greater  than  its  control  (and  generally  they  were  much  higher).  In  nine  of  the 
thirteen  experiments,  no  polyspermy  was  observed  among  100  eggs  in  the  control 
suspensions;  the  highest  control  level  was  4.7%.  (The  low  control  levels  ofpolyspermy 
in  Table  II  and  Figure  4  show  that  there  was  no  problem  with  overinsemination.) 
In  the  four  experiments  with  SOD  (the  protein  least  effective  at  2  mg/ml  in  inducing 
polyspermy),  the  highest  control  level  was  1.2%,  and  three  were  zero;  the  lowest 
level  with  SOD  was  2.1%,  and  its  associated  control  was  zero.  As  shown  in  Figure 
4,  there  was  a  high  probability  of  significance  of  the  effects  of  all  the  proteins  with 
the  possible  exception  of  SOD.  In  preliminary  experiments  ovalbumin  also  induced 
polyspermy  (1 1.3%;  control,  2.2%;  n  =  3). 


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FIGURE  4.  Induction  of  polyspermy  in  Arbacia  eggs  by  various  proteins,  all  at  2  mg/ml.  Sperm 
were  freshly  diluted  and  had  100%  fertilizing  capacity.  The  experimental  design  for  inducing  and  quan- 
tifying polyspermy  is  given  in  Materials  and  Methods  and  is  illustrated  in  Table  II.  The  values  are  means 
of  the  per  cent  polyspermic  eggs  (aberrant  cleavage)  of  cleaved  eggs  45-60  min  after  mixing  sperm  and 
eggs.  Error  bars  represent  SEMs;  numbers  of  determinations  are  also  shown.  P  is  the  level  of  significance 
of  difference  from  the  control,  i.e.  sperm  alone  (randomization  test  for  matched  pairs). 


PROTEIN  ACTIVATION  OF  ARBACIA  SPERM 


427 


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30 


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10 


•  — 
o  — 

•  — 

Protein 
cololase 
soybean  trypsin 
inhibitor 
gelatin 

_D_ 

2 

2 

1 

0 


001  0.1  I 

Protein  concentration :  mg/ml 


O 


FIGURE  5.  Induction  by  catalase,  SBTI,  or  gelatin  of  polyspermy  in  Arbacia  eggs  as  a  function  of 
the  concentrations  of  the  proteins  (mg/ml).  Sperm  were  freshly  diluted,  and  had  100%  fertilizing  capacity. 
The  experimental  design  is  given  in  Materials  and  Methods  and  is  illustrated  in  Table  II.  The  ordinate, 
"per  cent  polyspermy,"  is  the  per  cent  of  cleaved  eggs  which  were  polyspermic  (aberrant  cleavage).  Protein 
concentrations  on  the  abscissa  are  plotted  in  a  logarithmic  scale.  The  inset  shows  the  symbols  for  the 
proteins  and  n,  the  numbers  of  experiments  for  each. 


Figure  5  shows  the  dependence  of  induction  of  polyspermy  on  concentration 
for  three  of  the  proteins.  The  curves  describing  the  dependence  on  concentration 
expressed  as  w/v  are  similar  to  one  another  despite  a  difference  of  an  order  of 
magnitude  in  molecular  weight  between  catalase  and  SBTI  (see  Table  I).  The  results 
confirm  and  extend  the  recent  results  from  similar  experiments  (Schuel  et  al.,  1976; 
Coburn  et  al.,  1981;  Schuel  and  Schuel,  1981),  in  which  catalase  and  SBTI  induced 
polyspermy.  Figure  5  shows  that  these  two  proteins  induced  polyspermy  at  the  lowest 
concentration  tested  (5  Mg/ml),  nearly  as  low  as  the  concentration  (1.7  Mg/ml)  at 
which  most  of  the  proteins  could  reactivate  the  fertilizing  capacity  of  the  aged  sperm 
(Fig.  2).  The  results  are  in  conflict  with  studies  by  Coburn  et  al.  (1981)  and  Schuel 
and  Schuel  (1981),  in  which  a  failure  of  BSA  to  induce  polyspermy  was  reported. 
However,  Coburn  et  al.,  (1981)  reported  that  boiled  catalase  (concentration  not 
given)  induced  <20%  polyspermy  (and  therefore  presumably  some  polyspermy). 

DISCUSSION 

In  this  study  we  show  that  proteins  can  promote  activity  of  Arbacia  sperm.  The 
activities  measured  were  fertilizing  capacity,  motility,  and  polyspermy.  The  proteins 
stimulated  fertilizing  capacity  and  motility  of  inactive  sperm,  and  induced  poly- 
spermic fertilization  of  eggs  by  fresh  sperm. 

Brief  incubations  (6  minutes  or  less)  with  any  of  six  proteins  reactivated  aged 
sperm,  and  no  proteins  were  tested  which  were  ineffective.  The  diversity  of  the 
proteins  makes  clear  the  limited  specificity  of  their  effect:  two  are  enzymes  (CAT, 
SOD),  one  is  an  enzyme  inhibitor  (SBTI),  two  are  nonenzymatic  serum  proteins 
(BSA,  CFV),  and  one  (GEL;  boiled  collagen)  is  particularly  lacking  in  reactive  groups 
(Miller  and  Gay,  1982). 


428  P.   DUNHAM   ET  AL. 

The  dose-response  curves  for  activation  of  aged  sperm  (and  for  induction  of 
polyspermy,  though  only  3  proteins  were  tested)  show  that  the  concentration  de- 
pendence is  not  on  a  molar  basis,  but  on  concentration  as  weight/volume.  For 
example,  CAT  and  SOD  were  about  equally  effective  in  reactivating  aged  sperm  at 
17  Mg/ml  (Fig.  2),  though  their  molecular  weights  differ  by  nearly  an  order  of  mag- 
nitude. Similarly  CAT  and  SBTI  had  comparable  activities  in  inducing  polyspermy 
below  0.1  mg/ml  (Fig.  5).  Despite  this  evidence  for  limited  specificity,  in  most 
instances  the  proteins  were  active  in  promoting  both  functions  at  5  Mg/ml  or  less. 
These  various  considerations  make  difficult  the  task  of  deducing  the  mechanism  of 
action  of  the  proteins  on  sperm  function.  Prevention  of  binding  of  inhibitors  seems 
possible  but  not  likely:  gelatin  is  as  active  in  its  effects  on  sperm  function  as  the 
other  proteins,  but  lacks  reactive  groups  (gelatin  is  totally  lacking  in  cysteine  residues; 
Miller  and  Gay,  1982). 

Metal  chelators  can  delay  inactivation  of  sperm  (Rothschild  and  Tyler,  1954; 
Tyler  and  Tyler,  1966).  The  relative  affinity  of  such  a  chelator  as  ethylene  diamine 
tetracetate  (EDTA)  for  Cu++  (the  probable  inhibitor  of  sperm  in  sea  water;  Roths- 
child and  Tyler,  1954)  is  8-10  orders  of  magnitude  higher  than  for  Ca  and  Mg,  the 
prevalent  divalent  cations  in  sea  water  (logic  K^q  of  EDTA  for  Ca:  10.6;  for  Mg: 
8.8;  for  Cu:  18.7;  Martell  and  Smith,  1974).  Therefore,  even  though  the  concentra- 
tions of  Ca  and  Mg  in  sea  water  are  ~4  orders  of  magnitude  higher  than  the 
concentration  of  Cu,  EDTA  would  have  a  much  greater  effect  on  [Cu]  than  on  [Ca], 
and  could  thereby  influence  sperm  function. 

However,  it  is  unlikely  that  proteins  have  so  pronounced  an  effect.  Most  natural 
amino  acids  (except  cysteine)  have  about  the  same  affinity  for  Cu  as  EDTA  has  for 
Mg,  and  proteins  lacking  cysteine  have  a  much  lower  affinity  for  Cu  than  do  amino 
acids  (for  five  different  pentapeptides  the  Iog10  K^,  for  Cu  was  about  5.4;  Martell 
and  Smith,  1974),  and  as  stated  several  times  above,  gelatin,  lacking  cysteine  resi- 
dues, was  as  active  as  the  other  proteins  which  contain  reactive  groups,  in  its  effect 
on  sperm  function. 

Furthermore,  if  the  mechanism  of  reactivation  of  aged  sperm  by  proteins  is 
similar  to  their  mechanism  of  inducing  polyspermy,  then  chelation  of  heavy  metals 
cannot  be  the  sole  process  involved;  a  role  of  heavy  metals  in  the  block  to  polyspermy 
appears  unlikely. 

Interference  with  (or  binding  to)  inhibitory  substances  released  from  eggs  (e.g. 
fertilizin)  might  explain  induction  of  polyspermy,  but  cannot  explain  the  activation 
of  aged  sperm  which  have  not  been  in  contact  with  eggs  or  their  products. 

Two  recent  preliminary  studies  on  the  effects  of  bovine  serum  albumin  on  rodent 
sperm  suggest  a  role  for  proteins  in  capacitation,  i.e.  preparation  for  the  acrosome 
reaction  (Bavister,  1981;  Go  and  Wolf,  1981).  This  suggestion  may  or  may  not  be 
of  relevance  to  A r bad a  sperm. 

Whatever  the  mode  of  action  of  proteins  in  inducing  polyspermy,  our  results 
militate  against  the  suggestions  of  Coburn  el  al.  (1981)  and  Schuel  ei  al.  (1976)  of 
specific  enzymatic  or  enzyme  inhibitory  effects  based  on  induction  of  polyspermy 
by  CAT  and  SBTI,  since  serum  albumins  and  gelatin  are  also  effective  (Fig.  5). 

ACKNOWLEDGMENTS 

This  work  was  supported  by  grants  from  the  NIH  (AM  27851  to  PD  and  AM 
1 1949  to  GW)  and  the  NSF  (PCM  8002358  to  LN). 


PROTEIN   ACTIVATION  OF  ARBACIA   SPERM  429 

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Reference:  Biol.  Bull.  163:  431-437.  (December  1982) 


AN  ECHINODERM  VITELLARIA  WITH  A  BILATERAL 

LARVAL  SKELETON:  EVIDENCE  FOR  THE  EVOLUTION 

OF  OPHIUROID  VITELLARIAE  FROM  OPHIOPLUTEI 

GORDON   HENDLER 

Smithsonian  Oceanographic  Sorting  Center,  Smithsonian  Institution,  Washington,  D.  C.  20560 

ABSTRACT 

Ophionereis  annulata  (Le  Conte)  possesses  a  barrel-shaped,  yolky,  non-feeding 
vitellaria  larva  with  transverse  ciliary  bands.  However,  the  larva  develops  vestiges 
of  skeletal  structures  that  are  characteristically  present  in  feeding  ophiopluteus  larvae 
but  absent  from  vitellariae.  Thus,  it  is  evident  that  the  vitellaria  of  O.  annulata  is 
a  modified  ophiopluteus.  The  presence  of  a  pluteus-like  skeleton  in  a  vitellaria  larva 
is  suggestive  that  the  evolution  of  ophiuroid  larval  types  proceeds  in  a  gradual  fashion 
with  a  larval  skeleton  remaining  after  other  ophiopluteus  structures  are  lost.  Ophiu- 
roid vitellariae  have  apparently  evolved  from  ophiopluteus  larvae.  These  findings 
support  Mortensen's  (1921)  proposal  that  the  lecithotrophic  vitellaria  is  a  modified 
pluteus  and  contradict  the  hypothesis  (Fell,  1945;  Williams  and  Anderson,  1975) 
that  vitellaria  larvae  are  divergent  and  distinct  from  the  feeding  ophiopluteus  larvae. 

INTRODUCTION 

Johannes  Miiller  and  other  prominent  zoologists  of  the  19th  century  discovered 
that  each  class  of  living  echinoderms  (with  the  exception  of  the  crinoids)  has  a 
feeding  (planktotrophic)  larva  with  a  distinctive  body  plan:  the  holothuroid  auricu- 
laria,  the  asteroid  bipinnaria,  the  echinoid  echinopluteus,  and  the  ophiuroid  ophio- 
pluteus. Miiller  (1850)  also  described  a  yolky,  non-feeding  (lecithotrophic)  larva  of 
ophiuroids.  Because  of  its  shape  he  called  it  a  vermiform  (wurmformige)  larva;  this 
type  of  larva  was  later  renamed  a  vitellaria  (Fell,  1945). 

Certain  species  in  all  living  echinoderm  classes  have  yolky,  non-feeding,  larvae 
that  differ  strikingly  from  the  better  known  feeding  larvae.  The  crinoids  that  have 
been  reared  produce  yolky,  barrel-shaped  vitellariae  (also  called  doliolariae)  with 
four  or  five  transverse  bands  of  cilia.  Holothuroids  have  similar  vitellariae  (dolio- 
lariae), either  as  the  definitive  larva  or  as  a  secondary  larval  stage.  Ophiuroid  vi- 
tellariae look  very  much  like  the  barrel-shaped  crinoid  and  holothuroid  larvae  and 
generally  have  four  transverse  ciliary  bands. 

Fell  (1945)  introduced  the  term  "vitellaria"  for  the  larvae  which  he  considered 
a  divergent  series  possessing  yolky,  barrel-shaped  bodies  and  transverse  bands  of 
cilia.  Williams  and  Anderson  (1975)  drew  a  further  distinction  between  lecitho- 
trophic larvae  that  retain  vestiges  of  feeding  larva  structures  and  a  separate  group 
of  vitellaria  larvae  which  lack  any  vestiges  of  feeding  larva  structures  (such  as  a 
single  ciliary  band,  mouth,  anus,  larval  arms,  and  larval  skeleton).  In  their  view, 
reduced  larvae  such  as  Peronella  leseuri  echinoplutei  or  Amphiura  chiajei  ophio- 
plutei  are  manifestly  unlike  the  vitellaria  larvae  of  the  echinoid  Heliocidaris  ery- 
throgramma  or  the  ophiuroid  Ophioderma  brevispinum.  Prior  to  these  works,  how- 
ever, Mortensen  (1898,  1921)  treated  ophiuroid  vitellaria  larvae  as  a  variety  of 

Received  3  June  1982;  accepted  24  September  1982. 

431 


432  GORDON   HENDLER 

ophiopluteus.  A  consideration  of  the  vitellaria  of  Ophionereis  annulata  (Le  Conte) 
(Fig.  1A),  as  discussed  below,  supports  Mortensen's  contention  that  the  ophiuroid 
vitellaria  is  a  modified  ophiopluteus. 

MATERIALS  AND  METHODS 

I  collected  Ophionereis  annulata  at  a  depth  of  12  m  near  Taboguilla  Island  in 
the  Bay  of  Panama  (Central  America)  on  15  May  1975.  Specimens  were  taken  to 
the  Galeta  Marine  Laboratory  of  the  Smithsonian  Tropical  Research  Institute  on 
the  Carribbean  Coast  of  Panama.  A  female  ophiuroid,  held  in  the  laboratory  in  a 
fingerbowl  with  sea  water,  spawned  spontaneously  two  days  after  collection.  Its 
oocytes  were  fertilized  using  a  dilute  suspension  of  spermatozoa  from  the  dissected 
testes  of  a  male  specimen  of  O.  annulata. 

Temperature  in  the  laboratory  cultures  was  24-26  °C,  approximately  that  of  the 
water  at  the  collecting  site.  The  process  of  larval  development  in  these  cultures  is 
described  from  sketches  of  live  specimens,  from  fresh  squash  preparations  examined 
under  standard  and  polarized  illumination,  and  from  preserved  samples. 

RESULTS 

The  ova  of  Ophionereis  annulata  are  round,  0.24  mm  in  diameter,  and  pale 
yellow-green  to  yellow-brown.  They  were  denser  than  sea  water,  settling  to  the 
bottom  of  the  culture  vessel.  Within  1.5  h  after  fertilization  the  embryos  reached 
the  8-cell  stage,  and  blastulae  0.27  mm  in  diameter  developed  by  5  h.  Each  blastula 
almost  filled  the  vitelline  membrane  and  did  not  move  within  it. 

Swimming  larvae  were  found  near  the  bottom  of  the  culture  vessels  by  10  h  after 
fertilization,  and  by  1 2  h  some  larvae  swam  near  the  surface  of  the  water.  A  24-h 
gastrula  was  about  0.3 1  mm  long,  0.23  mm  wide,  and  somewhat  wedge  shaped  with 
a  blastopore  at  the  center  of  the  broadened  posterior  end.  It  swam  with  the  narrow 
end  foremost,  rotating  clockwise  around  the  long  axis  of  the  body. 

Several  important  changes  were  noted  36  h  after  fertilization.  The  blastopore 
was  no  longer  visible,  a  hydropore  penetrated  the  mid-dorsal  surface  of  the  larva, 
and  internally,  a  branching  hydrocoel  encircled  the  presumptive  oral  area. 

A  triradiate  level  skeleton  had  appeared  in  each  posterior  corner  of  the  larval 
body  by  34  h  (Fig.  IB).  In  the  36-h  larva,  the  pair  of  spicules  had  grown,  and  in 
specimens  38  h  old  the  three  branches  of  each  elongate  larval  skeletal  element  pre- 
sumably corresponded  to  the  body,  postoral,  and  posterolateral  rods  of  the  pluteus 
skeleton  (Fig.  1C).  The  more  complex  branching  pattern  of  the  48-h  larval  skeleton 
may  be  an  indication  of  the  formation  of  homologues  of  the  posterodorsal,  antero- 
lateral,  and  transverse  rods  of  the  ophiopluteus  skeleton  (Fig.  ID). 

A  pentaradiate  ophiuroid  rudiment  (i.e.  the  developing  adult  body),  on  the  mid- 
ventral  surface  of  the  38-h  larva,  below  the  branched  hydrocoel,  possessed  a  concave 
central  oral  area  and  tube  foot  buds.  The  entire  surface  of  the  38-h  larva  including 
the  ophiuroid  rudiment  was  ciliated.  Thickened,  more  densely  ciliated  ridges  were 
present  at  the  posterior  end  of  the  larval  body.  Apical  cilia  longer  than  the  body 
cilia  projected  from  the  anterior  end  of  the  larva. 

By  48  h  the  hydropore  was  no  longer  visible.  At  that  stage  the  larva  was  ap- 
proximately 0.42  mm  long  and  0.30  mm  wide.  The  larval  skeleton  was  displaced 
from  the  posterolateral  corners  of  the  larva,  possibly  through  the  growth  and  torsion 
of  the  ophiuroid  rudiment  preceding  metamorphosis.  Elements  of  the  adult  ophiu- 
roid skeleton  were  conspicuous  in  squash  preparations.  They  had  appeared  as  tri- 


OPHIUROID  VITELLARIA  SKELETON 


433 


FIGURE  1 .  (A)  62-hour  vitellaria  larva  of  Ophionereis  annulata  drawn  from  life;  c, ,  anterior  ciliated 
band  on  the  funnel-shaped  preoral  lobe;  c2  and  c3,  posterior  ciliated  bands  on  the  ophiuroid  rudiment; 
f,  triad  of  the  two  buccal  tube  feet  and  a  terminal  tube  foot  lying  between  the  epineural  folds;  a,  arm  tip 
of  the  ophiuroid  rudiment  containing  a  terminal  arm  plate.  B-D  are  photographs  of  squash  preparations 
of  specimens  viewed  with  polarized  illumination  that  makes  skeletal  elements  appear  white  and  soft 
tissues  of  the  larva  appear  dark:  s,  larval  skeleton;  t,  terminal  arm-plate  of  the  definitive  ophiuroid 
skeleton.  (B)  34-hour  larva  with  paired  posterolateral  rudiments  of  the  larval  skeleton.  The  hydrocoel  is 
a  scalloped  structure  at  the  center  of  the  larva.  (C)  38-hour  larva  with  thin,  slightly  branched  larval 
skeleton  and  five  triradiate  terminal  arm-plates.  Bulging  projections  at  the  posterior  end  of  the  larva  will 
develop  into  ciliated  bands.  (D)  48-hour  larva  with  branched  larval  skeleton  and  pentaradiate  terminal 
arm-plates.  The  preoral  lobe  and  ciliated  ridges  are  seen  in  silhouette.  Length  of  the  scale  is  0.10  mm. 


434  GORDON   HENDLER 

radiate  spicules  by  38  h,  and  by  42  h  they  formed  multibranched  terminal  arm  plates 
as  well  as  central,  radial,  and  oral  plates  of  the  disc  (Fig.  1C). 

By  48  h  the  preoral  lobe  of  the  vitellaria  formed  a  funnel  shape,  giving  the  larva 
a  distinctive  appearance  (Fig.  ID).  The  larva  had  only  three  ciliary  bands.  The 
absence  of  a  fourth  small  ciliary  band  on  an  elongation  of  the  preoral  lobe  distin- 
guishes O.  annulata  from  other  ophiuroid  vitellariae,  even  from  the  vitellaria  of 
Ophionereis  squamulosa  described  by  Mortensen  (1921). 

At  48  h  cilia  had  disappeared  from  the  ophiuroid  rudiment.  The  remainder  of 
the  larval  body  was  ciliated,  but  cilia  were  concentrated  along  the  transverse  ciliary 
bands.  As  the  ophiuroid  rudiment  matured,  cilia  disappeared  from  most  of  the  larval 
body,  and  by  62  h,  ciliation  was  restricted  to  well-defined  bands  encircling  the  larva 
(Fig.  1  A).  Ciliary  bands  on  the  72-h  larva  were  opaque  yellow-green,  but  other  areas 
of  the  larval  body,  particularly  the  preoral  lobe,  had  lost  their  yolky  opacity. 

The  tube  feet  of  62-h  larvae  were  papilliform,  and  by  72  h  the  three  tube  feet 
on  each  arm  of  the  ophiuroid  rudiment  were  capable  of  independent  movement. 
Tube  feet  at  the  tip  of  the  arm  protruded  from  within  the  terminal  arm  plate  and 
movements  of  the  paired  buccal  tube  feet  set  the  jaw  apparatus  in  motion. 

The  88-h  larva  was  about  0.42  mm  long  and  0.31  mm  wide,  approximately  the 
same  size  as  the  72-h  larva.  At  this  stage  there  was  scanty  ciliation  on  the  ventral 
surface  of  the  larvae,  but  lateral  ciliation  on  the  ciliary  bands  was  still  evident.  The 
ophiuroid  rudiment  appeared  opaque,  due  at  least  in  part  to  the  growing  density 
of  the  adult  skeleton  and  and  the  addition  of  new  skeletal  structures  such  as  teeth. 
Larval  skeletal  elements,  however,  were  completely  resorbed  by  88  h.  They  had 
reached  their  maximum  size  by  62  h,  and  over  the  next  26  h,  the  disappearing 
skeleton  remained  in  one  interradial  sector  of  the  ophiuroid  rudiment. 

The  newly  settled  Ophionereis  annulata  moved  by  propelling  the  disc  with  the 
distal  buccal  pair  of  tube  feet  and  the  first  pair  of  arm  spines.  The  tips  of  the  tube 
feet  bear  papillate  extensions,  much  like  the  juveniles  of  Amphioplus  abditus  dis- 
cussed by  Hendler  (1977).  Within  24  h  after  settlement,  portions  of  the  larval  body 
with  yellow-green  pigmentation  were  resorbed  and  the  locomotory  activity  and 
agility  of  the  juveniles  increased.  Within  8  days  after  settlement  the  stomach  of  the 
juvenile  formed  a  distinct  yellow  structure  within  the  disc.  It  is  not  known  whether 
the  pigmentation  of  the  gut  was  from  larval  yolk  or  ingested  food. 

DISCUSSION 

Fully  developed  ophioplutei  generally  have  four  pairs  of  larval  arms,  and  the 
abbreviated  pluteus  larvae  of  ophiuroids  constitute  a  continuous  reduction  series 
with  fewer  arms  than  normal  (Fell,  1945;  Hendler,  1975).  For  example,  Amphiura 
filiformis  has  three  pairs,  and  both  Amphiura  chiajei  and  Ophiothrix  oerstedii  have 
only  one  pair  of  larval  arms  (Mortenson,  1921;  Fell,  1945;Fenaux,  1963;  Mladenov, 
1979).  Although  the  latter  two  species  probably  do  not  feed,  they  are  clearly  reduced 
ophioplutei  that  retain  simplified  pluteus  arms,  skeleton,  and  ciliation.  There  are 
species  of  brooding  ophiuroids  (e.g.,  Axiognathus  squamatus  and  Ophionotus  hex- 
actis)  that  have  embryos  with  vestigial  larval  features,  and  other  brooders  have 
embryos  lacking  pluteus  or  vitellaria  characteristics  (e.g.,  Ophiomyxa  brevirima) 
(Mortensen,  1921;  Fell,  1941,  1946).  The  brooded  embryos  with  vestigial  ophio- 
pluteus  structures  have  been  considered  to  be  modified  ophioplutei  (Fell,  1946). 

Thus,  there  are  reduced  ophiuroid  larvae  which,  like  the  reduced  (and  secondary) 
larvae  of  asteroids,  holothuroids,  and  echinoids,  retain  certain  salient  vestiges  of  the 


OPHIUROID  VITELLARIA  SKELETON  435 

feeding  larva.  These  larval  types  would  seem  to  bear  no  clear  relation  to  the  vitellaria 
larvae  since  vitellariae  lack  even  vestigial  feeding  structures  and  are  characterized 
by  their  barrel-like  shape  and  multiple  transverse  ciliary  bands  that  are  used  solely 
for  locomotion,  and  not  for  feeding. 

It  is  therefore  surprising  that  Mortensen  (1898)  assigned  ophiopluteus  names  to 
ophiuroid  vitellariae,  evidently  believing  that  the  yolky  larvae  were  modified  feeding 
larvae.  Hamann  (1901)  objected  to  Mortensen's  nomenclature,  pointing  to  a  lack 
of  ophiopluteus  structures  such  as  larval  skeleton  in  the  vitellariae.  Later,  Mortensen 
(1921)  detected  irregular  calcareous  structures  in  the  vitellariae  of  the  ophiuroid 
Ophiolepis  cincta,  and  he  reiterated  that  the  vitellaria  was  a  reduced  ophiopluteus. 
The  larval  skeleton  of  Ophionereis  annulata  by  its  structure  and  its  position  in  the 
larva,  is  more  like  an  ophiopluteus  skeleton  than  are  the  spicules  of  O.  cincta  and, 
therefore,  provides  better  evidence  that  vitellariae  are  derived  from  a  feeding  larval 
stage  with  a  bilateral,  branched  larval  skeleton.  However,  this  deduction  assumes 
that  vitellaria  and  ophiopluteus  larval  skeletons  are  homologous. 

Compelling  evidence  for  homology  of  the  vitellaria  skeleton  with  the  ophioplu- 
teus skeleton  lies  in  the  fact  that  both  are  composed  of  branching,  rod-like  forms 
that  develop  at  the  posterolateral  corners  of  the  larval  body  and  that  are  resorbed 
during  metamorphosis.  Moreover,  the  only  echinoderm  larvae  with  skeletal  rods  are 
vitellariae  and  plutei,  and  pluteus  larval  rods  originate  in  a  manner  similar  to  the 
skeleton  of  0.  annulata.  It  is  highly  unlikely  that  the  form,  location,  and  ontogenesis 
of  larval  skeletons  would  be  duplicated  by  ophioplutei  and  vitellariae  if  the  skeletal 
elements  were  not  homologous. 

Assuming  that  the  skeletal  structures  of  vitellariae  and  ophioplutei  are  homol- 
ogous, is  there  additional  evidence  that  the  vitellaria  is  derived  from  the  ophioplu- 
teus? The  necessary  logical  framework  for  a  solution  to  this  question  was  devised 
by  Strathmann  (1974,  1978)  who  argued  that  lecithotropic  larvae  are  derived  from 
feeding  larvae.  The  most  persuasive  evidence  for  the  evolution  of  lecithotrophic 
larvae  from  planktotrophic  types  is  furnished  by  the  fact  that  such  a  change  requires 
a  reduction  rather  than  the  repeated  acquisition  of  extremely  complex  characters 
such  as  the  single  ciliary  band  feeding  mechanism  and  a  complete  larval  gut  (Strath- 
mann, 1974,  1978).  Furthermore,  some  traits  of  planktrophic  echinoderm  larvae 
are  unique  to  the  phylum,  whereas  lecithotrophic  larvae  with  transverse  ciliary  bands 
are  simple  forms  that  occur  in  unrelated  taxa,  indicating  convergence  (Jagersten, 
1972;  Strathmann,  1974). 

The  ophiopluteus  skeleton,  like  the  single  ciliary  band  feeding  mechanism,  is 
a  complex  structure,  presumably  more  readily  lost  than  evolved.  Thus,  as  suggested 
by  the  trend  of  progressive  reduction  in  size  and  complexity  of  the  larval  skeleton 
shown  in  ophioplutei  with  6,  4,  and  2  larval  arms,  it  might  be  expected  that  more 
highly  simplified  and  modified  ophioplutei  (e.g.  vitellariae)  could  retain  some  vestige 
of  an  ophiopluteus  larval  skeleton. 

As  already  mentioned,  the  same  reasoning  has  been  applied  in  treating  the 
relationship  between  ophioplutei  and  the  specialized  embryos  of  brooding  species 
such  as  Axiognathus  squamatus  and  Ophionotus  hexactis.  The  paired  skeletal  ele- 
ments in  these  embryos  are  regarded  as  vestiges  of  an  ophiopluteus  skeketon.  More- 
over, the  formation  of  coelomic  cavities  in  the  ophiuroid  vitellariae  also  appears  to 
be  a  simplification  of  the  process  of  coelomogenesis  in  the  ophioplutei  (Grave,  1916). 
Therefore,  I  regard  the  larval  skeleton  in  the  vitellaria  of  Ophionereis  annulata  as 
a  vestigial  rather  than  as  a  neomorphic  (i.e.,  newly  evolved)  structure. 

The  vitellaria  of  Ophionereis  annulata  provides  the  best  indication  that  the 


436  GORDON   HENDLER 

ophiuroid  vitellaria  evolved  from  an  ophiopluteus  form.  The  continuity  of  the  plank- 
totrophic,  reduced,  and  vitellaria  larval  forms  evidenced  by  the  skeleton  of  the  O. 
annulata  vitellaria  negates  the  distinction  drawn  by  Fell  (1945)  and  Williams  and 
Anderson  (1975)  between  vitellariae  and  feeding  larvae,  and  I  consider  these  various 
ophiuroid  larval  forms  to  be  homologous. 

The  presence  of  a  vestigial  larval  skeleton  in  Ophionereis  annulata  implies  that 
the  loss  of  feeding  larval  structures  during  the  evolution  of  yolky  larvae  may  be  a 
gradual  process.  Most  vitellaria  larvae  have  lost  the  larval  digestive  tract,  arms,  and 
single  ciliary  band,  as  well  as  the  larval  skeleton  of  the  ancestral  ophiopluteus  form. 
However,  the  retention  of  a  larval  skeleton  in  Ophionereis  annulata  (and  perhaps 
in  Ophiolepis  cincta)  suggests  that  the  larval  skeleton  may  be  lost  later  than  other 
larval  structures.  In  the  vitellaria  of  the  related  species  Ophionereis  squamulosa,  loss 
of  pluteus  larval  structures  is  complete.  Mortensen  (1921)  did  not  mention  a  larval 
skeleton  in  O.  squamulosa  and  I  have  reared  O.  squamulosa  (unpub.  obs.)  and 
found  no  trace  of  the  skeleton.  Therefore,  the  larva  ofO.  squamulosa  is  presumably 
a  more  advanced  form  than  the  larva  of  O.  annulata. 

ACKNOWLEDGMENTS 

I  thank  especially  Dr.  Richard  Strathmann,  and  Drs.  Daphne  Fautin  Dunn, 
Peter  Glynn,  Harilaos  Lessios,  David  Pawson,  John  Pearse,  Tyson  Roberts,  and  Ms. 
Maureen  Downey  and  Ms.  Tracey  Wolfe  for  discussing  with  me  some  of  the  ques- 
tions inspired  by  Ophionereis  annulata  vitellariae.  Comments  of  two  anonymous 
reviewers  were  also  very  helpful.  I  am  indebted  to  Dr.  Richard  Cloney,  who  prepared 
the  photographs  of  vitellaria  skeletons  in  this  paper  and  sacrificed  his  sunglasses  for 
microscopical  polarizing  elements.  Most  of  this  research  was  supported  by  a  Smith- 
sonian Institution  Walter  Rathbone  Bacon  Scholarship.  The  penultimate  draft  was 
written  while  crossing  the  Drake  Passage,  enroute  to  Palmer  Station,  Antarctica, 
aboard  the  NSF  research  vessel  Hero.  Part  of  the  research  reported  and  my  partic- 
ipation in  that  cruise  were  made  possible  by  Smithsonian  Institution  Fluid  Research 
Funds,  which  are  gratefully  acknowledged. 

LITERATURE  CITED 

FELL,  H.  B.  1941.  Probable  direct  development  of  some  New  Zealand  ophiuroids.  Trans.  Rov.  Soc. 

N.  Z  71:  25-26. 
FELL,  H.  B.  1945.  A  revision  of  the  current  theory  of  echinoderm  embryology.  Trans.  Rov.  Soc.  N.  Z. 

75:  73-101. 
FELL,  H.  B.  1946.  The  embryology  of  the  viviparous  ophiuroid  Amphipholis  squamatus  Delle  Chiaje. 

Trans.  Roy.  Soc.  N.  Z.  75:  419-465. 
FENAUX,  L.  1963.  Note  Preliminaire  sur  le  developpement  larvaire  de  Amphiura  chiajei  (Forbes).  Vie 

et  Milieu  14:  91-96. 
GRAVE,  C.  1916.  Ophiura  brevispina  II.  An  embryological  contribution  and  a  study  of  the  effect  of  yolk 

substance  upon  development  and  developmental  processes.  J.  Morphol.  27:  4 1 3-45 1 . 
HAMANN,  O.  1901.  Die  Schlangensterne.  Pages  751-956  in  Dr.  H.  G.  Bronn's  Klassen  und  Ordnungen 

des  Thier-Reichs,  H.  Ludwig,  Ed.,  Vol.  2  (3),  C.  F.  Winter' sche  Verlagshandlung,  Leipzig. 
HENDLER,  G.  1975.  Adaptational  significance  of  the  patterns  of  ophiuroid  development.  Am.  Zool.  15: 

691-715. 
HENDLER,  G.  1 977.  Development  ofAmphioplus abditus (Verrill)  (Echinodermata:  Ophiuroidea)  I.  Larval 

Biology.  Biol.  Bull.  152:  51-63. 

JAGERSTEN,  G.  1972.  Evolution  of  the  Metazoan  Life  Cycle.  Academic  Press,  New  York.  282  pp. 
MLADENOV,  P.  1979.  Unusual  lecithotrophic  development  of  the  Caribbean  brittle  star  Ophiothrix  oer- 

stedi.  Mar.  Biol.  55:  55-62. 
MORTENSEN,  TH.  1898.  Die  Echinodermenlarven  der  Plankton-Expedition  nebst  einer  systematischen 

Revision  der  bisher  bekannten  Echinodermenlarven.  Ergebnis.  Plankton-Exped.  Humboldt-Stift. 

2J:  1-118. 


OPHIUROID  VITELLARIA  SKELETON  437 

MORTENSEN,  TH.  1921.  Studies  on  the  Development  and  Larval  Forms  of  Echinoderms.  G.E.C.  Gad, 

Copenhagen.  261  pp. 
MULLER,  J.  1850.  Ueber  die  Larven  und  die  Metamorphose  der  Echinodermen.  Dritte  Abhandlung. 

Abhandl.  Konigl.  Preuss.  Akad.  Wiss.  Berlin.  1849:  35-72. 
STRATHMANN,  R.  R.  1974.  Introduction  to  function  and  adaptation  in  echinoderm  larvae.  Thalassia 

Jugoslavia  10:  321-339. 
STRATHMANN,  R.  R.   1978.  The  evolution  and  loss  of  feeding  larval  stages  of  marine  invertebrates. 

Evolution  32:  894-906. 
WILLIAMS,  D.  H.  C,  AND  D.  T.  ANDERSON.  1975.  The  reproductive  system,  embryonic  development, 

and  metamorphosis  of  the  sea  urchin  Heliocidaris  erythrogramma  (Val.)  (Echinoidea:  Echi- 

nometridae).  Ausl.  J.  Zool.  23:  371-403. 


Reference:  Biol.  Bull.  163:  438-452.  (December  1982) 


77V   VITRO  STUDIES  ON  THE  EFFECTS  OF  CELL-FREE  COELOMIC 

FLUID,  CALCIUM,  AND/OR  MAGNESIUM  ON  CLUMPING  OF 

COELOMOCYTES  OF  THE  SEA  STAR  ASTERIAS  FORBESI 

(ECHINODERMATA:   ASTEROIDEA) 

K.   KANUNGO 

Department  of  Biological  and  Environmental  Sciences,  Western  Connecticut  State  University. 

Danbury.  CT  06810 

ABSTRACT 

In  asteroid  echinoderms  the  loss  of  coelomic  fluid  due  to  injury  is  prevented  by 
the  clumping  of  coelomocytes  at  the  site  of  the  wound.  Plasma  (cell-free  coelomic 
fluid  =  CF)  coagulation  has  not  yet  been  demonstrated  in  these  animals.  An  in  vitro 
system  was  used  to  quantify  the  effects  of  CF,  Ca2+,  and/or  Mg2+  on  coelomocyte 
clumping  in  the  sea  star  Asterias  forbesi. 

The  results  show  that  the  coelomocytes  of  A.  forbesi  require  threshold  levels  of 
Ca2+  and/or  Mg2+  for  clumping  in  vitro,  and  these  levels  depend  on  whether  the 
ions  are  used  separately,  in  combination,  or  as  components  of  CF.  The  findings  also 
suggest  that  the  in  vitro  coelomocyte  clumping  is  mediated  by  a  factor  present  in 
CF  which  requires  Ca2+  and  Mg2+  to  be  effective.  A  two-phase  clumping,  consisting 
of  a  fast  phase  followed  by  a  slow  phase,  is  also  demonstrated. 

The  observed  biphasic  clumping  is  explained  by  the  existence  of  two  functional 
subpopulations  among  the  coelomocytes  which  differ  in  their  permeability  char- 
acteristics and  ability  to  establish  surface  adhesiveness  for  clumping.  Morphological 
identities  of  these  two  subpopulations  remain  to  be  ascertained. 

INTRODUCTION 

While  it  is  known  that  the  clumping  of  the  coelomocytes  occurs  as  a  means  of 
hemostasis  in  asteroid  echinoderms  (sea  stars)  (see  reviews  by  Endean,  1966;  Need- 
ham,  1970;  Belamarich,  1976;  Kanungo,  1982),  controversy  exists  as  to  the  type  of 
coelomocyte  and  the  mechanism  involved  in  such  cellular  clumping.  (In  this  paper 
the  terms  "aggregation"  and  "agglutination"  are  used  interchangeably  with  "clump- 
ing" of  coelomocytes  in  vivo  or  in  vitro,  and  the  term  "cell"  is  used  to  refer  to  the 
"coelomocyte.") 

Boolootian  and  Giese  (1958,  1959)  maintained  that  bladder  amoebocytes  trans- 
formed into  filiform  amoebocytes  which  then  agglutinated  to  form  plasmodial  clots 
in  eight  species  of  sea  stars  which  they  investigated.  The  filiform  stage  was,  therefore, 
viewed  as  a  precoagulant  phase.  Johnson  and  Beeson  (1966)  on  the  other  hand, 
reported  that  the  filiform  stage  was  not  required  to  initiate  or  to  maintain  coelomo- 
cyte clumps  in  the  sea  star  Patiria  miniata. 

In  analyzing  the  mechanism  of  of  coelomocyte  agglutination,  Boolootian  and 
Giese  (1959)  also  observed  that  the  agglutination  was  not  dependent  on  calcium  but 
on  the  formation  of  disulfide  linkages.  However,  Jangoux  and  Vanden  Bossche 

Received  30  April  1982;  accepted  24  September  1982. 

Abbreviations:  CF,  cell-free  coelomic  fluid  (plasma);  CMFSS,  calcium-  and  magnesium-free  salt 
solution;  Hepes,  N-2-hydroxyethylpiperazine  N-2-ethanesulfonic  acid;  NEM,  N-ethylmaleimide. 

438 


SEA   STAR  COELOMOCYTE  CLUMPING  439 

(1975)  reported  that  certain  amounts  of  calcium  were  required  to  induce  coelomo- 
cyte  clumping  in  Asterias  rubens. 

Factors  other  than  calcium  have  also  been  implicated  in  sea  star  coelomocyte 
clumping  in  vitro  (Boolootian  and  Giese,  1959;  Jangoux  and  Vanden  Bossche,  1975; 
Kanungo,  1982)  and  in  vivo  (Bang  and  Lemma,  1962;  Bang,  1970;  Reinisch  and 
Bang,  1971;  Reinische,  1 974).  These  studies  suggest  that  a  factor  released  at  the  time 
the  animal  is  wounded  or  challenged  with  foreign  materials  mediates  coelomocyte 
clumping.  A  factor  capable  of  inducing  clumping  in  the  coelomocytes  of  A.  forbesi 
has  been  isolated  from  the  coelomocytes  of  this  sea  star  (Prendergast  and  Suzuki, 
1970;  Prendergast  et  al.,  1974).  However,  the  existence  of  a  clotting  factor  in  the 
plasma  (coelomic  fluid  free  of  coelomocytes  =  CF)  of  sea  stars  has  not  yet  been 
demonstrated.  The  present  study  provides  some  experimental  evidence  for  the  ex- 
istence of  such  a  factor  in  the  CF  and  examines  the  role  of  calcium  and  magnesium 
in  agglutination  of  the  coelomocytes  of  A.  forbesi. 

MATERIALS  AND  METHODS 
Animals 

Asterias  forbesi  were  purchased  from  the  Marine  Biological  Laboratory,  Woods 
Hole,  Massachusetts.  They  were  held  in  the  laboratory  at  12°C  in  30-gallon  aquaria 
with  filtered,  recirculating,  continuously  aerated  sea  water  (salinity  30%o).  No  more 
than  nine  animals  were  kept  in  one  aquarium,  and  the  animals  (wet  weight  155- 
210  g)  were  used  within  ten  days  of  their  arrival  in  the  laboratory. 

Before  the  experiment  the  animals  were  screened  under  a  low  power  dissecting 
microscope  for  surface  wounds,  and  those  without  any  visible  wounds  or  abnor- 
malities were  used. 

Collection  of  coelomic  fluid 

Two  or  three  sea  stars  were  removed  from  the  holding  tank,  placed  in  a  pail 
containing  fresh  sea  water  at  room  temperature  (22°C),  and  held  there  for  about 
0.5  h. 

The  animal  was  blotted  with  a  soft  sponge  and  weighed.  It  was  then  held  upright 
to  allow  the  coelomic  fluid  to  accumulate  in  the  downward-hanging  arms.  When 
the  arms  were  visibly  swollen,  the  dermal  papullae  near  the  tip  of  the  swollen  arms 
were  abraded  with  a  razor  blade.  Coelomic  fluid  ( 1  ml)  was  allowed  to  drop  into 
a  1 5-ml  graduated  centrifuge  tube,  coated  inside  with  a  thin  layer  of  paraffin  (melting 
point  60°C),  which  held  9  ml  of  calcium-  and  magnesium-free  salt  solution  (CMFSS) 
containing  15  mM  ethylenediamine  tetracetic  acid  (EDTA).  CMFSS  was  prepared 
by  dissolving  the  following  components  in  a  liter  of  glass-distilled  water:  NaCl,  25.5 
g;  KC1,  0.8  g;  Na2SO4,  3.0  g;  glucose,  3.0  g;  and  Hepes  (N-2-hydroxy-ethylpiperazine 
N-2-ethanesulfonic  acid),  2.86  g.  The  pH  of  CMFSS  and  CMFSS-EDTA  solutions 
was  adjusted  to  7.4  with  NaOH.  The  solutions  were  filtered  through  presterilized 
0.22-^m  Millipore  filters  and  stored  in  sterile  containers  until  use. 

Preparation  of  coelomocyte  suspension 

Soon  after  collection  the  coelomic  fluid-CMFSS-EDTA  solution  was  mixed  by 
gentle  pipetting  several  times  through  a  Pasteur  pipette.  The  resulting  cell  suspension 
was  then  centrifuged  at  200  X  g  for  ten  minutes.  It  was  determined  by  light  mi- 
croscopy that  the  coelomocytes  thus  treated  did  not  lyse  or  suffer  visible  damage. 


440  K.   KANUNGO 

Almost  all  cells  in  the  suspension  were  separate  and  nonclumped.  The  supernatant 
was  discarded  and  the  cell  pellet  was  resuspended  in  fresh  CMFSS.  Cell  counts  were 
made  using  a  hemocytometer.  Only  nonclumped  cells  were  counted,  and  the  cell 
density  was  adjusted  to  about  106  cells/ml.  Usually  coelomocytes  from  several  an- 
imals were  pooled  to  run  replicate  experiments. 

The  test  system 

The  experimental  system  we  used  to  assess  the  sensitivity  of  coelomocytes  to 
CF  and  to  Ca2+  and/or  Mg24"  is  as  follows.  Cell  suspension  in  CMFSS  (10  ml)  was 
placed  in  a  25-ml  Erlenmeyer  flask,  the  inside  of  which  was  coated  with  a  thin  layer 
of  paraffin.  This  reaction  flask  was  then  placed  in  a  shaker  water  bath  at  20°C  and 
agitated  at  50-60  revolutions  per  minute.  A  count  of  nonclumped  cells  was  made 
immediately  after  the  cell  suspension  was  placed  in  the  flask.  This  count,  taken  at 
time  zero,  is  referred  to  as  the  initial  count.  A  test  substance(s)  (CF,  CaCl2,  and/or 
MgCl2)  was  then  added  (at  various  concentrations)  to  the  suspension.  Stock  solutions 
(for  CF  see  below)  of  the  test  salts  were  prepared  in  deionized  water.  Reagent  grade 
chemicals  were  used  in  all  experiments.  The  volume  of  a  test  substance(s)  added 
to  the  reaction  flask  did  not  exceed  1%  of  the  volume  of  cell  suspension  in  the  flask. 
The  concentration  of  CF  in  the  suspension  is  expressed  as  /ul  CF/ml  CMFSS,  whereas 
those  of  CaQ2  and/or  MgCl2  are  expressed  in  millimolar  (mA/)  units  of  Ca2+  and 
Mg2+  assuming  100%  dissociation  of  the  salts  in  the  test  system.  Control  systems 
were  prepared  and  incubated  in  exactly  the  same  manner  as  the  experimentals  but 
contained  appropriate  volumes  of  deionized  water  in  place  of  a  test  solution. 

Nonclumped-cell  counts  were  made  at  five-minute  intervals  for  a  total  experi- 
mental period  of  30  min.  A  significant  decrease  in  nonclumped-cell  number  during 
an  experiment  was  considered  to  be  due  to  clumping  of  cells  since  other  factors  that 
could  cause  such  a  decrease  in  our  in  vitro  system  were  eliminated  (see  below).  Thus 
a  concentration  of  a  substance  in  the  test  system  producing  a  significant  decrease 
in  nonclumped-cell  count  during  the  period  of  the  experiment  is  referred  to  as 
"clumping  concentration,"  and  one  that  did  not  produce  such  a  decrease  is  termed 
a  "nonclumping  concentration."  Microscopic  observations  on  samples  taken  from 
the  reaction  flasks  were  conducted  along  with  the  cell  counts  to  determine  if  clump- 
ing of  coelomocytes  had  actually  occurred. 

Tests  for  cell  attachment  and/or  lysis 

Since  coelomocyte  attachment  to  the  vessel  wall  and/or  lysis  of  these  cells  could 
cause  a  reduction  in  nonclumped-cell  counts  in  our  in  vitro  system,  the  following 
experiments  were  performed  to  determine  if  these  possibilities  existed  in  our  test 
system. 

(a.)  A  portion  of  cell  suspension  containing  a  clumping  concentration  of  CF 
(10  Ml/ml)  or  of  Ca2+  and  Mg2+  (0.23  and  0.12  mM,  respectively)  was  placed  on 
paraffinized  slides  and  incubated  in  a  humidified  chamber  at  room  temperature  for 
30  min.  The  suspension  was  drained  off  and  the  slide  was  inspected  under  a  com- 
pound microscope  for  possible  cell  attachment,  (b.)  The  cell  suspension  containing 
the  above  mentioned  clumping  concentration  of  CF  or  Ca24  +  Mg2""  was  incubated 
for  30  min  in  a  manner  similar  to  other  experimental  systems  described  under  the 
test  system.  After  the  incubation  the  cell  suspension  was  centrifuged,  and  the  pellet 
was  resuspended  in  CMFSS  containing  10  rriA/ N-ethylmaleimide  (NEM).  A  non- 
clumped-cell count  was  made  to  determine  if  the  initial  nonclumped-cell  number 
was  restored. 


SEA   STAR  COELOMOCYTE  CLUMPING  441 

Preparation  and  assay  of  normal,  dialyzed,  and  heated  CF 

Normal.  Coelomic  fluid  was  collected  in  a  precooled,  paraffinized  centrifuge  tube 
by  abrading  the  animals  as  described  above.  It  was  then  centrifuged  at  200  X  g  for 
10  min.  The  cell  pellet  was  discarded,  and  the  supernatant  was  filtered  through  a 
sterilized  0.22-)um  millipore  filter  and  stored  at  —  20°C  in  sterile  containers. 

Experiments  were  performed  with  different  concentrations  of  CF  ranging  from 
5  to  10  )ul/ml  (at  graded  concentration  intervals  of  CF)  to  establish  a  cutoff  point 
between  nonclumping  and  clumping  concentrations  of  CF. 

Dialyzed.  Fifty  ml  of  normal  CF  was  dialyzed  against  500  ml  of  CMFSS  for  48 
h  at  4°C  with  constant  stirring.  CMFSS  was  changed  five  times  during  the  48-h 
period.  At  the  end  of  this  period,  CF  was  sterilized  by  filtration  and  stored  as  dis- 
cussed above. 

The  clumping  effectiveness  of  dialyzed  CF  was  tested  by  adding  the  CF  at  10 
jul/ml  to  the  test  system.  This  concentration  was  chosen  because  experiments  with 
normal  CF  suggested  this  was  a  clumping  concentration. 

Heated.  Normal  CF  was  heated  for  15  min  at  100°C  then  cooled,  sterilized  by 
filtration,  and  stored  as  described  above.  This  CF  was  also  assayed  at  10  ^1/ml  for 
its  clumping  effectiveness. 


Experiments  with  Ca~+  and/or 

Calcium  and  magnesium  were  assayed  in  absence  of  CF  by  adding  various 
concentrations  of  these  ions,  independently  of  each  other,  to  the  test  system.  From 
these  experiments  nonclumping  and  clumping  concentrations  for  Ca2+  and  Mg24 
were  established.  Similarly,  nonclumping  and  clumping  concentrations  of  Ca2+ 
+  Mg24  were  determined  by  assaying  the  two  ions  in  combination  in  the  test  system. 

"Reconstitution  "  experiments 

These  experiments  were  designed  to  test  the  clumping  ability  of  dialyzed  CF  in 
the  presence  of  Ca2+  and  Mg2+.  Two  series  of  experiments,  were  performed,  (i.) 
initially  dialyzed  CF  at  10  jil/ml  (a  clumping  concentration  for  normal  CF)  was 
added  to  the  test  system.  The  system  was  then  incubated  for  30  min  after  which 
Ca2+  and  Mg24  were  added  to  yield  concentrations  of  0.  1  mA/  and  0.6  mA/,  re- 
spectively. These  specific  ion  concentrations  were  used  because  normal  CF  when 
added  to  the  test  system  at  a  concentration  of  10  /ul/ml  yields  0.  1  mA/  Ca2+  and  0.6 
mA/  Mg2+  (see  below  for  determination  Ca24  and  Mg2+  in  CF).  Subsequent  to  the 
addition  of  the  divalent  cations  in  the  entire  system  was  further  incubated  for  another 
30-min  period.  Nonclumped-cell  counts  were  made  at  5-  or  10-min  intervals  from 
the  beginning  of  the  experiment  to  the  end  of  the  second  30-min  incubation  period. 
(ii.)  The  sequence  of  addition  of  dialyzed  CF  and  Ca24  +  Mg2+  to  the  test  system 
was  reversed.  First  Ca2+  and  Mg24  were  added  to  obtain  concentrations  of  0.1  mA/ 
and  0.6  mA/,  respectively.  The  system  was  then  incubated  for  30  min  following 
which  dialyzed  CF  at  a  concentration  of  10  yul/ml  was  added.  Subsequently,  the 
entire  system  was  further  incubated  for  another  30-min  period.  Cell  counts  were 
made  during  the  entire  60-min  incubation  period  as  described  in  (i).  In  these  two 
series  of  experiments  the  initial  time  of  reconstitution  refers  to  the  time  when  the 
final  ingredient(s)  (dialyzed  CF  or  the  divalent  cations)  was  (were)  added  to  the  test 
system. 


442  K.   KANUNGO 

Coelomocyte  viability  tests 

After  the  experiment,  viability  of  coelomocytes  was  determined  by  two  separate 
methods:  Trypan  Blue  exclusion  method  (Phillips,  1973),  and  visual  observation  of 
coelomocyte  attachment  and  spreading  on  a  glass  surface.  For  the  latter  test  a  sample 
of  postexperimental  cells  was  placed  on  a  clean  glass  slide  and  observed  under  a 
phase  contrast  microscope.  Since  only  live  coelomocytes  can  attach  to  a  surface  and 
spread  by  extending  petaloid  pseudopodia  (formed  by  bladder  coelomocytes  which 
constitute  over  90%  of  the  total  coelomocyte  population  [Kanungo,  1982]),  those 
that  attached  and  spread  their  pseudopodia  were  considered  viable. 

Ca2+,  Mg2+,  and  osmolality  determinations 

Normal  and  dialyzed  CF  were  analyzed  on  a  flame  photometer  (Coleman  Model 
51,  Coleman  Instruments,  Perkin-Elmer  Corp.,  Maywood,  IL)  for  their  Ca2+  con- 
tents while  their  Mg2+  contents  were  determined  using  an  atomic  absorption  spec- 
trophotometer  (Perkin-Elmer  Model  560,  Perkin-Elmer  Corp.,  Maywood,  IL.) 

The  osmolalities  of  various  solutions  were  determined  using  an  Advanced  Dig- 
imatic  Osmometer  (Advanced  Instruments,  Inc.,  Needham  Heights,  MA).  The  in- 
strument operates  on  freezing  point  depression  principle  and  gives  a  readout  in 
milliosmoles/kg. 

Statistical  analysis  and  calculations 

Paired  t  tests  were  applied  to  compare  cell  counts  at  each  time  interval  with  that 
at  zero  time.  Data  were  considered  significant  at  the  95%  confidence  level. 

In  case  where  the  number  of  nonclumped  cells  is  expressed  as  %  of  the  initial 
number,  linear  regression  was  used  to  determine  the  best  fit  lines  (except  where 
indicated  otherwise). 

The  percent  clumping  was  calculated  by  subtracting  the  mean  cell  count  at  a 
given  time  point  from  that  at  time  zero  (or  at  time  30  min  in  reconstitution  ex- 
periments) and  taking  this  difference  as  percent  of  the  corresponding  initial  count. 


RESULTS 

The  assumption  that  a  decrease  in  the  number  of  single  coelomocytes  in  our  in 
vitro  system  was  not  due  to  attachement  and/or  lysis  of  cells  but  due  to  clumping 
must  be  justified,  for  if  this  assumption  is  not  valid  the  results  obtained  by  using 
this  system  would  be  meaningless. 

The  osmolalities  of  CMFSS  and  CF  were  933  and  955  mOsmol/kg,  respectively. 
They  were,  therefore,  considered  here  as  isoosmotic  for  all  practical  purposes.  Thus, 
the  coelomocytes  could  not  have  been  osmotically  stressed  even  though  the  coelomic 
fluid  was  diluted  10X  with  CMFSS  during  collection.  Almost  all  cells  remained 
viable  during  the  experimental  period  as  judged  from  post-experimental  cell 
viability  tests. 

Cell  attachment  and/or  lysis 

Microscopic  observations  showed  that  the  coelomocytes  did  not  attach  to  par- 
affinized  slides.  However,  the  same  cells,  when  placed  on  clean  glass  slides,  adhered 
to  the  glass  surface,  extended  petaloid  pseudopodia,  and  subsequently  underwent 
transformation  from  bladder  to  filiform  type. 


SEA  STAR  COELOMOCYTE  CLUMPING  443 

TABLE  I 

Changes  in  the  number  of  nonclumped  coelomocytes  of  (he  sea  star  Asterias  forbesi  in  the  test  system 
(cell  suspension  in  CMFSS)  and  after  exposure  to  clumping  concentrations  of  CF  or  Ca2+  +  Mg2+ 
for  30  min  in  vitro  followed  by  exposure  to  10  mM  NEM. 

Number  (x  ±  SD  X  10^4/ml)  of  nonclumped  coelomocytes 


Time 
(min) 

CMFSS 

CF(  10  Ml/ml) 

0.23  mM  Ca2+ 
+  0.12  mMMg2+ 

n+  =  9 

n  =  6 

n  =  6 

0 

104.00  ±  11.52 

115.00  ±8.17 

125.83  ±  20.45 

5 

103.78  ±  18.53 

— 

— 

10 

101.00  ±  23.36 

— 

— 

15 

107.00  +  22.46 

— 

— 

20 

100.89  ±  15.79 

— 

— 

25 

108.11  ±  21.38 

— 

— 

30 

104.56  ±  18.01 

70.83  ±  8.86* 

70.83  ±  10.57* 

+  NEM  +  NEM 

45  —  109.17  +  4.49  123.33  +  32.30 

*  Significant  at  95%  confidence  interval  in  paired  comparison  /  tests  between  the  indicated  mean 
(x)  and  that  at  time  zero. 

+  n  =  number  of  experiments. 


There  was  also  no  significant  decrease  in  the  number  of  nonclumped  cells  during 
the  30-min  experimental  period  when  the  cells  were  suspended  in  CMFSS  (Table 
I)  or  in  diluted  CMFSS  as  in  control  flasks.  However,  the  possibility  existed  that  a 
test  substance  in  clumping  concentration  in  the  system  might  cause  attachment  of 
the  coelomocytes  to  the  vessel  (cell  lysis  does  not  occur  in  CF  or  in  Ca2+  and  Mg2+) 
and  thus  produce  a  decrease  in  nonclumped-cell  count.  This  is  discounted  by  the 
results  given  in  Table  I  which  show  that  after  induction  of  clumping  the  initial 
number  could  be  restored  if  the  total  cell  population  in  the  system  was  resuspended 
in  NEM.  Thus,  the  contention  that  the  observed  decrease  in  the  number  of  coe- 
lomocytes in  our  test  system  was  due  to  clumping  and  not  due  to  attachment  and/ 
or  lysis  is  fully  justified. 

Effects  ofCF 

Normal  CF.  CF  of  A.  forbesi  contains  on  the  average  10  mM  Ca2+  and  60  mM 
Mg2+.  In  our  test  system  the  number  of  nonclumped  coelomocytes  did  not  decrease 
significantly  from  that  of  the  initial  number  when  the  cells  were  suspended  in  CMFSS 
containing  CF  at  concentrations  equal  to  or  lower  than  7  ^1/ml  (Table  II).  Increasing 
the  CF  concentration  to  7.5  /ul/ml  or  higher,  however,  resulted  in  significant  decrease 
in  nonclumped-cell  number  (Table  II).  The  clumps  produced  by  CF  ranged  from 
2-cell  to  5-cell  aggregates. 

The  overall  reduction  in  cell  counts  produced  by  CF  at  concentrations  of  7.5 
^1/ml  and  10  /ul/ml  in  a  30-min  period  were  22%  and  33%,  respectively  (Fig.  1). 
Both  reductions  were  significantly  different  from  the  corresponding  counts  at  time 
zero  (Table  II).  The  greatest  reduction  in  nonclumped  cell  counts  was  produced 
during  the  first  five-min  period  when,  on  the  average,  a  19%  clumping  was  observed. 
However,  in  the  next  25-min  period  the  two  CF  concentrations  produced  different 
patterns  of  clumping.  Although  a  3%  increase  was  observed  with  the  CF  concen- 


444 


K.   KANUNGO 


TABLE  II 

Effects  of  various  concentrations  and  treatment  of  CF  on  the  number  of  none-lumped  coelomocytes  of 
the  sea  star  Asterias  forbesi  at  different  time  intervals  under  in  vitro  conditions. 

Number  (x  ±  SD  X  l(T4/rnl)  of  nonclumped  coelomocytes 
Concentration  of  CF  (^l/ml)  in  test  flasks 


Time 
(min) 

5 
(n  = 

5) 

7 
(n  = 

5) 

7.5 
(n  =7) 

10 
(n  = 

9) 

10  (Dialysed) 
(n  =  8) 

10  (Heated) 
(n  =  8) 

0 

104.00  ± 

10.18 

102.20  ± 

12.06 

102.43  ± 

6.70 

112.11  ± 

9.22 

118.13  ± 

16.76 

1  19.00  ±  22.96 

5 

107.20  ± 

10.00 

95.80  ± 

13.36 

84.43  ± 

7.73* 

88.11  ± 

8.56* 

119.38  ± 

15.30 

117.83  ±  23.44 

10 

101.40  ± 

10.95 

92.20± 

20.54 

— 

89.89  ± 

17.34* 

113.75  ± 

9.27 

121.38  ±  40.73 

15 

— 

86.20  ± 

16.44 

81.43  ± 

7.73* 

84.22  ± 

14.33* 

111.88  ± 

18.86 

117.00  ±  34.45 

20 

97.20  ± 

16.36 

91.00  ± 

21.95 

83.43  ± 

8.52* 

82.33  ± 

18.22* 

117.50± 

19.69 

110.50  ±  29.83 

25 

97.20  ± 

12.25 

88.60  ± 

21.21 

82.86  ± 

7.38* 

80.00  ± 

14.51* 

107.50  ± 

19.36 

107.13  ±  25.79 

30 

97.40  ± 

8.16 

97.80  ± 

23.10 

78.14  ± 

10.25* 

72.67  ± 

9.52* 

113.13  ± 

10.88 

111.25  ±  28.34 

*  Significant  at  95%  confidence  interval. 


tration  of  7.5  ^1/ml  during  this  25-min  period,  this  was  not  significantly  different 
from  the  19%  clumping  produced  in  the  initial  period.  (The  regression  line  through 
these  time  points  is,  therefore,  horizontal  in  Fig.  1.)  On  the  other  hand,  a  significant 
increase  of  14%  over  the  initial  19%  was  observed  with  a  CF  concentration  of  10 
Ail/ml  during  the  same  25-min  period. 

Dialyzed  and  heated  CF.  The  osmolality  of  dialyzed  CF  was  933  mOsmol/kg, 
and  such  CF  did  not  contain  any  detectable  amount  of  Ca:*  or  Mg:+.  The  number 
of  nonclumped  coelomocytes  did  not  decrease  significantly  with  the  addition  of  10 


0      10     20     30     40     50     60 
MINUTES 

FIGURE  1.  Effects  of  cell-free  coelomic  fluid  (CF),  Ca2+,  and/or  Mg2+  on  clumping  of  coelomocytes 
of  Asterias  forbesi  in  vitro.  The  number  of  replicate  experiments  (n)  performed  in  each  category  is  shown 
in  Tables  II  and  III  except  those  of  the  "reconstitution"  experiments.  O  and  •  for  CF  concentrations 
of  7.5  Ml/ml  and  10  /*l/ml,  respectively.  A  and  •  for  0.45  mATCa2+  and  0.23  mAf  Ca2+  +  0.12  mA/Mg2+, 
respectively.  A  for  0.75  mA/  Mg2+.  Reconstitution  experiments  (see  text  for  details):  D,  initial  addition 
of  dialyzed  CF  (n  =  10);  ®,  initial  addition  of  Ca2+  +  Mg2+  (n  =  8).  Symbols  in  (  )  represent  the 
corresponding  mean  cell  counts  that  are  not  significantly  different  from  those  at  time  zero,  or,  in  "re- 
constitution"  experiments,  from  those  at  30  min. 


SEA   STAR  COELOMOCYTE  CLUMPING 


445 


of  dialyzed  or  heated  CF  to  the  system,  even  though  this  concentration  was 
well  above  the  minimal  clumping  concentration  of  normal  CF  (7.5  /til/ml) 
(Table  II). 


Effects  ofCa2+  and/or 

There  was  no  significant  decrease  in  nonclumped-cell  numbers  with  addition 
of  0.23  mM  Ca2+  or  0.50  mM  Mg2+  to  the  test  system  (Table  III).  Similarly,  no 
reduction  in  cell  number  was  observed  when  0.  1  mM  Ca2+  +  0.6  mM  Mg2+  were 
added  to  the  system.  However,  addition  of  0.23  mMCa2+  +  0.12  mMMg2+,  or  0.45 
mMCa2+  produced  a  reduction  of  45%  in  a  30-min  period,  whereas  0.75  mMMg2+ 
reduced  the  cell  number  by  about  32%  from  the  initial  during  the  same  period  (Fig. 
1).  In  addition,  the  greatest  amount  of  reduction  was  achieved  during  the  first  5- 
min  period  when  on  the  average  a  30%  decrease  in  nonclumped-cell  counts  was 
effected  with  the  above  concentrations  of  divalent  cations  (except  with  0.75  mM 
Mg2+).  During  the  next  25-min  period  a  reduction  of  about  15%  in  nonclumped- 
cell  number  was  observed  with  0.23  mMCa2+  +  0.12  mM  Mg2+  and  with  0.45  mM 
Ca2+.  In  Figure  1  ,  one  line  is  drawn  through  this  25-min  period's  time  points  because 
the  calculated  regression  lines  through  the  data  points  for  the  clumping  concentra- 
tions of  Ca2+  +  Mg2+  and  Ca2+  are  very  close  to  each  other.  It  is  therefore  reasonable 
to  conclude  that  coelomocyte  clumping  requires  higher  concentrations  of  Ca2+  or 
Mg2+  when  these  ions  are  used  individually  than  when  they  are  used  together  and 
that  the  divalent  cations  act  synergistically  in  regard  to  clumping. 

With  0.75  mM  Mg2+  no  significant  decrease  in  nonclumped-cell  count  occurred 
during  the  initial  5-min  period  (Fig.  1).  The  large  standard  deviation  associated  with 
the  mean  suggests  that  there  were  excessive  variations  among  the  replicate  counts 
taken  at  the  end  of  the  initial  5-min  period  (Table  III). 

Coelomocyte  clumping  pattern  with  "reconstituted"  CF 

The  reconstitution  experiments  showed  a  decline  in  nonclumped  cells  by  about 
40%  in  30  min  after  reconstitution.  In  Figure  1  the  line  through  these  time  points 
is  drawn  by  estimation  since  the  regression  lines  through  respective  data  points  with 


TABLE  III 

Effects  ofCa2+,  Mg2+,  and  Ca2+  +  Mg2+  on  the  number  of  nonclumped  coelomocytes  of  the  sea  star 
Asterias  forbesi  at  different  time  intervals  under  in  vitro  conditions. 

Number  (x  ±  SD  X  l(T4/ml)  of  nonclumped  coelomocytes 


Cone.  (mM)  ofCa2+ 


Cone.  (mM)  of  Mg2 


Cone.  (mM)  of  Ca2+  +  Mg2+ 


Time 
(min) 

0.23 
(n  =  6) 

0.45 
(n  =  6) 

0.50 
(n  =  10) 

0.75 
(n  =  5) 

0.10  +  0.60 
(n  =  8) 

0.23  + 
(n  = 

0.12 
11) 

0 

107.67  ±  21.57 

113.29  ± 

9.66 

99.10± 

10.79 

112.20  ± 

5.77 

118.13  ± 

14.35 

122.73  ± 

17.21 

5 

99.67  ±  25.73 

78.43  ± 

11.76* 

96.30  ± 

24.15 

101.00  ± 

22.00 

113.75  ± 

13.64 

88.91  ± 

10.89* 

10 

94.33  ±  21.88 

76.43  ± 

10.73* 

88.80  ± 

22.18 

95.40  ± 

11.52* 

1  20.00  ± 

9.35 

80.00  ± 

11.26* 

15 

104.67  ±  25.77 

72.71  ± 

12.59* 

— 

89.80  ± 

13.86* 

113.75  ± 

15.56 

79.82  ± 

9.29* 

20 

104.33  ±  22.89 

65.57  ± 

10.07* 

91.08  ± 

25.29 

— 

11  2.50  ± 

15.00 

78.27  ± 

15.90* 

25 

104.33  ±  20.14 

66.86  ± 

14.61* 

89.50  ± 

19.87 

84.80  ± 

5.27* 

118.13  ± 

15.80 

68.64  ± 

8.97* 

30 

118.33  ±  29.53 

60.14  ± 

15.85* 

85.30  ± 

19.64 

74.20  ± 

6.65* 

113.13  ± 

14.98 

70.18  ± 

11.15* 

*  Significant  at  95%  confidence  interval. 


446  K.    KANUNGO 

these  two  sets  of  experiments  are  close  to  each  other.  The  reconstitution  experiments 
also  reveal  the  following  (ref.  Fig.  1):  (i.)  The  sequence  of  addition  of  dialyzed  CF 
or  the  divalent  cations  to  the  system  does  not  alter  the  extent  of  clumping  after 
reconstitution  as  judged  from  the  closeness  of  points  in  Figure  1.  (ii.)  The  time 
course  of  clumping,  for  the  1 5-min  period  following  reconstitution,  with  reconsti- 
tuted CF  is  different  from  that  with  normal  CF.  In  reconstitution  experiments  a  lag 
period  was  evident  when  no  significant  reduction  in  nonclumped-cell  numbers  was 
observed  until  15  min  after  reconstitution.  (The  two  points  corresponding  to  40  min 
period  in  Figure  1  are  not  significantly  different  from  zero  clumping  observed  at  the 
time  of  reconstitution.)  (iii.)  The  extent  of  clumping  at  the  end  of  30  min  after 
reconstitution  (60  min  from  time  0)  was  7%  higher  than  that  with  normal  CF  at 
10  ^1/ml  during  a  similar  clumping  period.  This  shows  that  the  addition  of  the 
equivalent  amount  of  divalent  cations  to  dialyzed  CF  or  vice  versa  restores  the 
clumping  effectiveness  of  dialyzed  CF.  Furthermore,  it  indicates  a  synergism  between 
the  divalent  cations  and  the  CF.  (iv.)  Since  clumping  could  be  achieved  with  the 
addition  of  10  /zl/ml  of  normal  CF  but  not  with  the  same  concentration  of  dialzyed 
CF  or  equivalent  concentrations  of  Ca24  and  Mg2+,  it  is  clear  that  CF-mediated 
coelomocyte  clumping  in  our  system  is  due  to  a  factor(s)  present  in  CF.  (v.)  From 
the  conclusions  stated  in  (iii)  and  (iv)  above,  it  follows  that  the  clumping  factor(s) 
present  in  CF  requires  Ca2+  and  Mg2+  to  produce  clumping  of  coelomocytes,  and 
in  the  absence  of  these  divalent  cations  the  factor(s)  is(are)  ineffective  as  a  clumping 
agent(s). 

DISCUSSION 

The  results  indicate  that  coelomocytes  clump  when  suspended  in  CMFSS  con- 
taining clumping  concentrations  of  CF,  Ca2+,  and/or  Mg2+.  The  failure  of  the  coe- 
lomocytes to  clump  when  suspended  in  CMFSS,  or  in  CMFSS  containing  CF  below 
7.5  Ml/ml  (Table  II),  0.23  mM  Ca2+,  0.50  mM  Mg2+,  or  0.1  mM  Ca2'  +  0.6  mM 
Mg2+  (Table  III),  demonstrates  that:  (i)  the  clumping  of  these  cells  is  dependent  on 
the  presence  of  Ca2+  and/or  Mg2+  and  a  clumping  factor(s)  in  the  suspending  me- 
dium; and  (ii)  a  minimum  concentration  of  Ca2+  and/or  Mg2^  in  the  medium  is 
necessary  for  clumping  to  occur.  The  necessary  concentration  of  Ca2^  and/or  Mg2^ 
for  clumping  is  also  dependent  on  whether  or  not  the  ions  are  used  with  CF,  and 
whether  they  are  used  separately  or  in  combination. 

Since  the  agglutination  of  hemostatic  cells  in  many  animals,  including  mam- 
malian platelets,  is  dependent  on  the  presence  of  Ca2+  and  Mg2+  in  the  medium 
(see  review  by  Belamarich,  1976;  Massini,  1977),  it  is  not  surprising  to  find  that  the 
coelomocytes  of  A.  forbesi  require  these  ions  for  clumping  in  vitro.  However,  the 
finding  of  Boolootian  and  Giese  (1959)  that  clumping  of  the  coelomocytes  of  8 
species  of  sea  stars  (which  do  not  include  A.  forbesi)  is  independent  of  Ca2+  warrants 
critical  examination  in  the  light  of  the  present  results.  The  authors  drew  this  con- 
clusion because,  in  their  system,  sea  star  coelomocytes  clumped  in  the  presence  of 
EDTA.  In  our  collection  system,  which  also  contained  EDTA,  the  coelomocytes 
remained  separate  and  nonclumped.  The  species  difference,  while  it  could  be  a 
factor,  is  an  unlikely  explanation  for  this  difference  in  results. 

Thus  the  reason  for  coelomocyte  clumping  in  the  collection  system  of  Boolootian 
and  Giese  must  be  sought  in  the  technique  used  by  the  authors  rather  than  in  the 
species  difference.  They  collected  0.9  ml  of  coelomic  fluid  in  0. 1  ml  of  EDTA 
solution  and  used  2  mM  and  13  mM  EDTA  at  pH  7.6  in  their  final  collection 
mixture.  We  used  EDTA  at  a  final  concentration  of  12.75  mM  at  pH  7.4.  It  is 


SEA   STAR  COELOMOCYTE  CLUMPING  447 

unlikely  that  the  pH  difference  would  account  for  the  diametrically  opposite  results 
obtained.  However,  it  is  possible  that  a  certain  amount  of  Ca2+  and/or  Mg2+  was 
left  unchelated  in  the  system,  and  these  free  ions  might  have  produced  clumping. 
Thus,  it  becomes  essential  to  discuss  the  kinetics  of  chelation  of  these  divalent 
cations  with  EDTA  in  the  system  used  by  Boolootian  and  Giese. 

The  amount  of  EDTA  present  in  the  total  1  ml  mixture  of  Boolootian  and  Giese 
was  either  2  X  10~3  or  13  X  1CT3  mmoles.  The  authors  did  not  report  the  concen- 
trations of  Ca2+  and  Mg2+  in  the  coelomic  fluid  of  the  sea  stars  they  used.  Thus,  for 
purposes  of  present  calculations,  we  have  used  the  data  reported  by  Binyon  (1972) 
which  show  that  the  average  concentrations  of  Ca2+  and  Mg2+  in  the  coelomic  fluid 
of  four  species  of  sea  stars  (Astropecten  sp.,  Solaster  endica,  Asterias  vulgaris,  and 
Marthaster  glacialis)  are  10.8  mM  and  46.4  mM,  respectively.  Accordingly,  the 
respective  amounts  of  these  ions  present  in  the  coelomic  fluid-EDTA  mixture  of 
Boolootian  and  Giese  were  9.7  X  10~3  mmoles  of  Ca2+  and  41.8  X  10~3  mmoles 
of  Mg2+.  Although  the  amounts  of  free  Ca2+  and  Mg2+  in  the  coelomic  fluid  would 
be  less  than  the  total  amounts  (because  of  association  with  other  ions),  it  is  reasonable 
to  use  the  figures  for  the  total  amounts  of  these  ions  in  calculations  of  their  chelation 
with  EDTA.  The  formation  constants  for  Ca-EDTA  and  Mg-EDTA  at  pH  7  are  2.5 
X  107  and  2.5  X  105,  respectively  (Kolthoff  et  al.,  1969).  Therefore,  when  both  ion 
species  are  present  in  an  equal  mole  ratio,  Ca-EDTA  is  expected  to  be  formed 
preferentially  over  Mg-EDTA  in  a  ratio  of  100:1.  However,  in  the  above  coelomic 
fluid  mixture  the  mole  ratio  of  Ca2+:Mg24  1:4.3.  This  would  produce  Ca-EDTA 
and  Mg-EDTA  in  an  approximate  ratio  of  23:1.  Since  one  mole  of  EDTA  binds 
one  mole  of  divalent  cation,  it  follows  that  the  maximum  amount  of  divalent  cation- 
EDTA  complex  that  could  be  formed  in  the  mixture  is  either  2  X  10"3  mmoles  or 
13  X  10"3  mmoles,  depending  on  the  concentration  of  EDTA  used. 

With  a  binding  ratio  of  23:1  and  with  9.7  X  10  3  mmoles  of  Ca2+  present  in  the 
mixture,  it  can  be  easily  calculated  that  2  X  10~3  mmoles  of  EDTA  could  bind  only 
1.91  X  10"3  mmoles  of  Ca2+.  Therefore,  7.8  X  10~3  mmoles  of  Ca2+  and  all  the  Mg2+ 
would  be  left  uncomplexed  in  the  mixture.  However,  with  1  3  mM  EDTA,  all  Ca24 
present  in  the  mixture  would  be  chelated,  while  38.5  X  10  3  mmoles  of  Mg2+  would 
be  left  uncomplexed.  The  concentration  of  this  uncomplexed  Mg2+  in  the  mixture 
is  about  51X  more  than  the  clumping  concentration  (0.75  mM)  reported  here. 
Obviously,  when  the  system  contained  2  mM  EDTA,  uncomplexed  Ca2+  had  pro- 
duced clumping.  When  it  contained  13  mM  EDTA,  uncomplexed  Mg2+  was  prob- 
ably responsible  for  clump  induction.  Further,  our  data  show  that  Mg2+,  in  the 
absence  of  Ca2+,  is  not  capable  of  maintaining  clumps,  provided  the  cell  suspension 
in  anticoagulant  solution  is  stirred  properly.  Collecting  coelomic  fluid  from  the 
animals  in  a  syringe,  as  was  done  by  Boolootian  and  Giese,  probably  did  not  provide 
sufficient  mixing  of  the  fluid.  This  insufficient  mixing  together  with  uncomplexed 
Ca2+  or  Mg2+  produced  clumping  in  their  collection  system.  Hence,  their  charac- 
terization that  the  clumping  of  sea  star  coelomocytes  is  independent  of  Ca2+  is 
unwarranted.  The  present  findings  and  those  of  Jangoux  and  Vanden  Bossche  (1975) 
clearly  demonstrate  that  the  clumping  of  the  sea  star  coelomocytes  depends  on  the 
presence  of  Ca2+  and/or  Mg2+  in  the  medium. 


Clumping  pattern  with  CF,  Ca:+  and/or 

The  time  course  of  clumping  in  the  presence  of  CF  at  10  jul/ml,  0.23  mM  Ca24 
+  0.12  mM  Mg2+,  or  0.45  mM  Ca2+  is  biphasic  with  an  initial  phase  occurring 
during  the  first  5-min  period  and  a  second  phase  following  (Fig.  1).  This  biphasic 


448  K.    KANUNGO 

mode  of  clumping,  however,  was  not  evident  in  two  cases.  The  second  phase  clump- 
ing was  absent  with  CF  concentration  of  7.5  yul/ml,  while  no  initial  phase  could  be 
discerned  with  0.75  mM  Mg2+  (Fig.  1). 

Further,  the  data  presented  in  Figure  1  reveal  that  the  degree  of  clumping  in  the 
initial  phase  was  variable  and  increased  from  19%  with  CF  to  33%  with  Ca24  and 
Ca2+  +  Mg2+  (except  with  0.75  mM  Mg2+).  Evidently  not  all  coelomocytes  that  were 
potentially  capable  of  clumping  formed  clumps  with  CF  concentration  of  7.5  or  10 
/ul/ml.  It  is  interesting  to  note  that  the  two  clumping  concentrations  of  CF  produced 
identical  clumping  in  the  initial  phase.  Similarly,  0.45  mM  Ca2+  and  0.23  mM  Ca2"* 
+  0.12  mM  Mg2+  also  produced  identical  clumping  in  the  initial  phase  (Fig.  1)  even 
though  they  differed  in  their  Ca2+  concentrations  by  a  factor  of  2.  Since  Mg24 
potentiates  the  clumping  action  of  Ca2+,  a  lower  Ca2+  concentration  in  the  presence 
of  Mg2+  could  produce  clumping  identical  to  that  with  a  higher  Ca2+  concentration 
in  the  absence  of  Mg2+.  Identical  clumping  rates  observed  with  0.23  mM  Ca24 
+  0.12  mM  Mg2+  and  0.45  mM  Ca2+  might  be  coincidental  and  not  necessarily 
indicative  of  maximal  clumping  for  the  initial  phase.  Variations  in  cell  counts  among 
replicate  experiments  which  resulted  in  abolition  of  the  initial  phase  in  the  case  of 
0.75  mM  Mg2+  could  have  occurred  if  the  individual  cells  forming  clumps  were  not 
adhered  to  each  other  firmly.  The  "loose"  clumps  would  dissociate  easily  and  pro- 
duce large  variations  in  nonclumped-cell  counts.  This  implies  that  with  0.75  mM 
Mg2+  it  takes  longer  for  the  coelomocytes  to  develop  "stickiness"  and,  therefore, 
more  time  is  required  to  form  "tight"  clumps  in  vitro. 

Sponge  cells  suspended  in  solutions  containing  EDTA  suffer  some  damage  and 
are  inhibited  from  clumping,  and  this  effect  can  be  reversed  by  supplying  proper 
amounts  of  Ca2+  and/or  Mg2+  (Humphreys,  1963).  EDTA  inhibition  of  Limulus 
amoebocyte  aggregation  was  reversed  completely  by  adding  24  mM  Mg2+  or  Limulus 
"serum"  at  1 :20  dilution,  but  reversal  was  incomplete  with  32  mM  Ca2+  (Kenney 
el  al.,  1972).  Our  results  show  that  coelomocytes  of  A.  forbesi,  which  have  been 
inhibited  from  clumping  with  EDTA  during  collection  and  centrifugation,  resume 
their  clumping  activity  at  a  faster  pace  if  immediately  suspended  in  CMFSS  con- 
taining clumping  concentrations  of  CF,  Ca2+  +  Mg2+,  or  Ca2+  than  they  do  if  im- 
mediately suspended  in  CMFSS  containing  a  clumping  concentration  of  Mg2+. 

During  the  second  phase,  although  the  overall  extent  of  clumping  increased  in 
a  linear  fashion  during  a  25-min  period,  the  rate  of  clumping  was  slower  than  that 
in  the  initial  phase.  Further,  CF  concentration  of  10  n\/m\,  0.23  mM  Ca2+  +0.12 
mM  Mg2+,  and  0.45  mM  Ca2+  produced  a  clumping  rate  of  6%  per  10  min  in  the 
second  phase,  suggesting  that  the  second  phase  clumping  occurs  independently  of 
the  initial  phase.  This  conclusion  is  also  supported  by  the  results  with  CF  at  a 
concentration  of  7.5  n\/m\  which  did  not  show  any  significant  clumping  in  the 
second  phase  while  producing  a  19%  clumping  in  the  initial  phase.  In  addition,  a 
constant  clumping  rate  during  the  second  phase  in  contrast  to  variable  rates  in  the 
initial  phase  suggests  that  the  mechanism  for  coelomocyte  clumping  are  different 
for  each  phase. 

Clumping  lag  with  reconstituted  CF 

The  delay  in  initial  clumping  in  reconstitution  experiments  could  have  been 
produced  by  the  prolonged  stay  of  the  coelomocytes  in  dialyzed  CF  or  nonclumping 
concentrations  of  Ca24  +  Mg2+.  Had  EDTA  produced  any  damage  to  the 
coelomocytes  during  collecting  and  centrifugation  it  could  only  have  been  accen- 
tuated by  not  returning  the  cells  to  a  medium  containing  proper  amounts  of  Ca2+ 


SEA   STAR  COELOMOCYTE  CLUMPING  449 

and/or  Mg2+.  Consequently,  prolonged  inhibition  of  clumping  under  these  condi- 
tions would  require  longer  recovery  time  and  produce  a  lengthy  lag  period  before 
clumping.  This  reasoning,  while  it  explains  the  delay  in  initial  clumping,  also  implies 
that  clumping  is  brought  about  by  the  "stickiness"  of  the  coelomocytes  and  that  a 
nonclumping  environment  impairs  the  development  of  this  "stickiness." 

That  the  divalent  cation  chelators,  EDTA  and  EGTA,  may  affect  cell  adhesion 
by  removing  materials  from  cell  surfaces  has  been  postulated  for  different  cell  types 
(Weiss,  1960;  Curtis,  1973;  Moscona,  1973).  Nobel  (1970)  has  also  expressed  similar 
views  with  regard  to  the  effects  of  EDTA  at  pH  6.0  on  the  aggregation  of  the 
coelomocytes  of  the  sea  cucumber  Cucumaria  frondosa. 

The  coelomocyte-clumping  factor  in  CF 

The  results  presented  here  strongly  suggest  the  existence  of  a  coelomocyte  clump- 
ing factor  in  CF  which  requires  Ca2+  and  Mg2+  to  be  effective.  That  the  factor  is 
nondialyzable  and  heat  labile  is  also  indicated  by  the  results.  It  might  be  argued  that 
dialyzing  CF  against  CMFSS  had  removed  not  only  Ca2+  and  Mg2+  but  also  other 
constituents,  such  as  trace  elements  and  low  molecular  weight  organic  compounds, 
from  CF.  The  removal  of  these  other  substances  could  affect  clumping.  Although 
the  argument  is  reasonable,  it  is  unlikely  that  these  components  exerted  any  effect 
on  clumping  of  the  coelomocytes.  The  restoration  of  clumping  with  reconstituted 
CF  would  not  have  been  possible  if  components  other  than  Ca2+  and  Mg2+  had  any 
appreciable  effect  on  clumping. 

Source  and  nature  of  clumping  factor(s) 

Two  possible  sources  of  the  clumping  factor(s)  exist.  The  factor(s)  could  have 
been  released  (i)  by  the  injured  tissue,  and/or  (ii)  from  the  coelomocytes  during 
collection  of  the  coelomic  fluid  and  the  preparation  of  the  CF.  Extracts  of  echi- 
noderm  tissues  have  been  shown  to  cause  coelomocyte  clumping  (Donnellon,  1938; 
Bookhout  and  Greenberg,  1940;  Davidson,  1953;  Boolootian  and  Giese,  1959).  That 
an  extract  prepared  from  coelomocytes  can  mediate  coelomocyte  clumping  in  the 
sea  star  has  also  been  demonstrated  (Bang  and  Lemma,  1962;  Bang,  1970).  The 
latter  observation  has  gained  strength  by  the  isolation  and  characterization  of  a 
clumping  factor  from  the  coelomocytes  ofA.forbesi  (Prendergast  and  Suzuki,  1970; 
Prendergast  and  Liu,  1976).  According  to  these  authors,  the  factor  is  a  basic  protein 
with  a  molecular  weight  of  approximately  38,000  daltons.  The  nondialyzability  and 
the  heat  labile  nature  of  the  factor(s)  reported  here  would  also  indicate  that  it  (they) 
is  (are)  a  protein(s).  However,  further  experimental  work  is  needed  to  determine  the 
exact  nature  and  the  source  of  the  clumping  factor(s). 

Mechanism  of  cellular  clotting 

Clotting  of  the  coelomic  fluid  in  echinoderms  is  achieved  by  the  agglutination 
of  the  coelomocytes.  Thus,  an  in  vitro  analysis  of  the  mechanism  of  cellular  aggre- 
gation (agglutination  or  clumping)  is  equated  with  the  analysis  of  the  mechanism 
of  clotting. 

Since  Geddes  ( 1 880)  first  documented  the  cellular  clotting  in  echinoderms  many 
investigators  have  provided  useful  information  on  the  subject  (see  review  by  Kan- 
ungo,  1982).  However,  except  for  the  work  of  Boolootian  and  Giese  (1959),  none 
of  the  reports  provide  experimental  studies  on  the  clotting  itself.  The  coelomocytes 
of  all  echinoderms  form  clumps  in  vitro  (Endean,  1966;  Johnson  and  Beeson,  1966; 


450  K.   KANUNGO 

Johnson,  1969;  Bang,  1970;  Chien  el  aL  1970;  Noble,  1970;  Fontaine  and  Lambert, 
1977;  Bertheussen  and  Seljelid,  1978;  Kaneshiro  and  Karp,  1980;  Kanungo,  1982). 
In  asteroid  echinoderms,  the  predominant  type  of  coelomocyte  is  the  bladder  amoe- 
bocyte,  which  takes  part  in  clotting  (Johnson  and  Beeson,  1966;  Bang,  1970;  Kan- 
ungo, 1982).  Transformation  of  bladder  to  filiform  amoebocytes,  which  was  thought 
to  be  a  prerequisite  for  cellular  clotting  in  sea  stars  (Boolootian  and  Giese,  1958, 
1959),  has  since  been  disputed  by  Johnson  and  Beeson  (1966)  and  Kanungo  (1982). 
Also,  the  contention  of  Boolootian  and  Giese  (1959)  that  the  cellular  clotting  in 
asteroid  echinoderms  does  not  require  Ca2"  is  no  longer  tenable  in  the  light  of  the 
present  findings  and  those  of  Jangoux  and  Vanden  Bossche  (1975). 

Our  results  clearly  demonstrate  the  requirement  of  Ca2^  and/or  Mg2+  for  clump- 
ing of  the  coelomocytes  in  vitro.  Further,  it  is  also  shown  that  in  the  presence  of  CF 
(which  contains  the  clumping  factor)  the  requirement  for  these  divalent  cations  for 
cellular  clumping  in  vitro  is  less  than  it  is  without  CF.  Taken  together  these  findings 
suggest  that  the  clumping  factor(s)  alters  the  permeability  of  the  cell  membrane  to 
divalent  cations  in  a  way  which  increases  the  influx  of  these  ions.  As  a  result,  the 
intracellular  concentrations  of  Ca24  and  Mg2+  increase  to  levels  at  which  clumping 
becomes  possible.  In  other  words,  the  coelomocyte  clumping  depends  on  the  intra- 
cellular rather  than  the  extracellular  concentrations  of  these  ions.  This  is  not  unusual 
in  light  of  the  second  messenger  role  played  by  Ca2  +  in  coordinating  diverse  cellular 
activities  in  many  cell  types  (Rasmussen,  1970;  Berridge,  1975),  including  mam- 
malian platelets  (Massini,  1977).  The  above  hypothesis  also  predicts  that  in  the 
absence  of  the  clumping  factor,  a  higher  concentration  of  Ca2*  and/or  Mg24  in  the 
medium  would  be  required  to  establish  a  concentration  gradient  that  would  favor 
a  greater  influx  of  these  ions  which  in  turn  would  cause  clumping.  The  present 
results  substantiate  this  hypothesis  because  clumps  could  be  formed  with  CF  con- 
centration at  7.5  jul/ml  which  contains  0.075  mA/Ca24  and  0.45  mA/  Mg2+,  but  in 
the  absence  of  CF  clumping  could  not  be  effected  even  at  concentrations  of  0. 1  mM 
Ca24  +  0.6  mM  Mg2+. 

Biphasic  clumping  and  its  implications 

A  two-stage  coelomocyte  clumping  has  been  reported  in  the  holothurian.  Cuc- 
umaria  frondosa,  by  Fontaine  and  Lambert  (1977).  The  authors  contended  that  the 
initial  fast  aggregating  stage  was  brought  about  by  the  transitional  cells  which  were 
present  in  the  coelomic  fluid  before  it  was  withdrawn  from  the  animal,  but  the 
second  slow  phase  was  due  to  the  transformation  of  the  bladder  amoebocytes  to  the 
transitional  form  which  occurs  at  a  slower  pace  in  vitro. 

It  is,  therefore,  conceivable  that  the  biphasic  clumping  reported  here  is  due  to 
two  functional  cell  populations  (a  fast  reacting  population  and  a  slow  reacting  one) 
which  exist  among  the  coelomocytes  of  A.  forbesi. 

The  cells  in  the  two  groups  probably  differ  in  their  permeability  characteristics 
and  their  ability  to  establish  surface  properties  for  clumping.  The  fast  reacting  cells 
become  ''sticky"  faster  than  the  slow  ones  in  the  presence  of  a  clumping  stimulus. 
Whether  these  two  populations  of  coelomocytes  differ  in  their  morphological  char- 
acteristics is  not  kno  vn.  Current  investigations  on  intercellular  adhesion  implicate 
cell  surface  glycoproteins  (Roseman,  1974;  Oppenheimer,  1977,  1979)  and  lectins 
(Brown  and  Hunt,  1978;  Rosen  and  Kaur.  1979)  in  generating  sites  for  mutual 
adhesion  of  cells  in  a  variety  of  cells  systems.  Similar  studies  using  the  coelomocytes 
of  echinoderms  would  provide  useful  information  for  elucidating  the  mechanism 
of  cellular  clumping  in  these  animals. 


SEA  STAR  COELOMOCYTE  CLUMPING  451 

ACKNOWLEDGMENTS 

I  thank  Dr.  Jack  Levin  of  the  Department  of  Medicine,  The  Johns  Hopkins 
University  School  of  Medicine  and  Hospital,  Baltimore,  Maryland,  for  reading  the 
manuscript  critically  and  offering  valuable  suggestions.  My  thanks  also  go  to  Mar- 
garet Dawson  and  Dr.  Frederick  Thurberg  of  the  National  Marine  Fisheries  Service, 
and  Milford  Laboratory,  Milford,  Connecticut,  for  the  use  of  the  Flame  Photometer, 
the  Atomic  Absorption  Spectrophotometer,  and  the  Advanced  Digimatic  Osmom- 
eter,  and  to  David  Harrison  and  Dr.  Susan  Maskel  of  Western  Connecticut  State 
College,  Danbury,  Connecticut  for  their  technical  help  in  various  phases  of  the  work. 

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Reference:  Biol.  Bull.  163:  453-464.  (December  1982) 


MALE  PRONUCLEAR  DEVELOPMENT  IN  STARFISH  OOCYTES 
TREATED  WITH    1-METHYLADENINE 

FRANK  J.   LONGO1  *   AND  ALLEN   W.   SCHUETZ2 

1  Department  of  Anatomy,  University  of  Iowa,  Iowa  City,  IA  52242,  ^Department  of  Population 

Dynamics,  John  Hopkins  School  of  Hygiene,  Baltimore.  MD  21205,  and  the 

'  '2  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

ABSTRACT 

Light  and  electron  microscopic  observations  were  carried  out  in  order  to  examine 
the  relation  between  male  pronuclear  development  and  the  state  of  "cytoplasmic 
maturation"  acquired  by  starfish  oocytes  under  the  influence  of  1-methyladenine 
(1-MA).  Alterations  were  not  apparent  in  the  germinal  vesicles  or  incorporated 
sperm  nuclei  of  inseminated  immature  Asterias  eggs  for  up  to  5  hours  in  the  absence 
of  1-MA.  With  the  addition  of  1-MA  dramatic  changes  occurred  in  the  germinal 
vesicle  and  ooplasmic  region  associated  with  incorporated  sperm  nuclei.  These  were 
followed  by  alterations  in  the  sperm  nucleus  leading  to  the  development  of  a  male 
pronucleus.  Pronuclear  development  in  Asterias  eggs  inseminated  at  the  germinal 
vesicle  stage  and  then  treated  with  1-MA  differed  from  that  described  for  other 
organisms.  Aside  from  the  dilation  of  its  perinuclear  cisterna,  the  sperm  nuclear 
envelope  persisted  intact  throughout  development.  Dispersion  of  condensed  chro- 
matin  occurred  simultaneously  throughout  the  whole  of  the  sperm  nucleus.  These 
results  suggest  that  factors  necessary  for  pronuclear  development  do  not  exist  in  the 
ooplasm  of  immature  starfish  oocytes  but  arise  following  dispersal  of  germinal  vesicle 
contents  into  the  cytoplasm. 

INTRODUCTION 

The  eggs  of  most  animals  initiate  meiotic  maturation  prior  to  ovulation,  become 
arrested  at  a  specific  stage  of  meiosis,  and  resume  maturation  after  insemination. 
Although  eggs  may  be  experimentally  manipulated  to  ferilize  prematurely,  i.e.,  at 
an  earlier  stage  of  meiosis,  investigations  with  the  ova  of  a  number  of  different 
organisms  have  indicated  that  germinal  vesicle  breakdown  is  a  prerequisite  in  es- 
tablishing a  condition  of  cytoplasmic  maturation  which  supports  the  transformation 
of  a  fertilizing  spermatozoon  into  a  male  pronucleus  (Skoblina,  1974,  1976;  Hirai, 
1976;  Katagiri  and  Moriya,  1976;  Thadani,  1979;  Balakier  and  Tarkowski,  1980; 
Hirai  el  al,  1981). 

Germinal  vesicle-intact  (immature)  starfish  oocytes,  induced  to  mature  by  ovar- 
ian hormone  (1-methyladenine;  1-MA),  develop  normally  when  fertilized  (Kanatani 
and  Shirai,  1967;  Schuetz  and  Biggers,  1967;  Kanatani  et  al,  1969).  Germinal  ves- 
icle-intact oocytes  may  also  be  inseminated  and  subsequently  treated  with  1-MA 
to  induce  germinal  vesicle  breakdown  (Cayer  et  #/.,  1975;  Schuetz,  1975;  Schuetz 
and  Longo,  1981).  That  the  onset  of  germinal  vesicle  breakdown  can  be  controlled 
by  exogenous  substances  in  starfish  eggs  provides  a  means  of  studying  nucleocy- 
toplasmic  interactions  during  male  pronuclear  development  and  the  role  of  1-MA 
in  fertilization  and  the  onset  of  development. 

Received  24  May  1982;  accepted  7  September  1982. 

*  Author  to  whom  correspondence  should  be  addressed  (at  Univ.  of  Iowa). 

453 


454 


F.   J.   LONGO  AND  A.   W.   SCHUETZ 


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PRONUCLEAR  DEVELOPMENT  IN  ASTERIAS  455 

The  present  light  and  electron  microscopic  study  examines  the  relation  between 
processes  of  fertilization  and  the  state  of  "cytoplasmic  maturation"  acquired  by 
oocytes  under  the  influence  of  1-MA.  For  this  purpose  observations  were  designed 
to  analyze  sperm-egg  interactions  in  fertilized,  intact  germinal  vesicle  oocytes  of  the 
starfish,  Asteriasforbesi,  before  and  after  exposure  to  1-MA.  Brief  accounts  of  these 
observations  have  been  published  previously  (Schuetz  and  Longo,  1979,  1981). 

MATERIALS  AND  METHODS 

Germinal  vesicle-intact  oocytes  were  obtained  from  ripe  Asteriasforbesi  as  de- 
scribed by  Longo  et  al.  (1982).  Oocytes  were  fertilized  with  a  0.1%  suspension  of 
sperm  and  samples  were  fixed  at  varying  intervals  up  to  5  hours  postinsemination. 
At  forty-five  minutes  postinsemination  a  portion  of  this  suspension  was  treated  with 
1-MA  (1  Mg/ml)  and  samples  were  taken  at  varying  intervals  and  prepared  for  light 
and  electron  microscopy  as  previously  described  (Longo  et  al,  1982).  A  second 
suspension  of  oocytes  was  treated  with  1-MA  (1  Mg/ml)  to  induce  meiotic  matu- 
ration; these  oocytes  are  referred  to  as  maturing  ova.  Approximately  30  minutes 
later  the  eggs  were  fertilized  and  sampled  at  periodic  intervals  up  to  90  minutes 
postinsemination. 

RESULTS 

Within  5  minutes  of  gamete  mixing,  sperm  were  seen  within  immature  and 
maturing  ova,  usually  located  at  the  base  of  the  fertilization  cone  (Fig.  1 ).  By  this 
time  the  cortical  granule  reaction  was  completed  and  a  fertilization  membrane  sur- 
rounded the  inseminated  egg  (Figs.  1,  2).  All  of  the  immature  starfish  oocytes  ex- 
amined in  this  study  were  polyspermic  (Schuetz  and  Longo,  1981);  eggs  that  had 
undergone  germinal  vesicle  breakdown  prior  to  insemination  were  monospermic. 
A  more  detailed  ultrastructural  account  of  male  and  female  pronuclear  development 
and  association  in  monospermic,  maturing  Asterias  eggs  is  the  subject  of  a  subse- 
quent report;  light  microscopic  observations  have  been  presented  by  Hirai 
et  al.  (1981). 

The  fertilization  cone,  through  which  the  sperm  nucleus  passed  during  its  in- 
corporation, was  larger  in  germinal  vesicle  oocytes  than  in  maturing  eggs.  When  the 
fertilization  cone  achieved  its  maximum  dimensions  (at  about  5  minutes  postin- 
semination) in  germinal  vesicle-intact  eggs  it  extended  approximately  2.5  /nm  from 
the  oocyte  surface  and  was  about  1  /um  in  diameter  at  its  base.  Morphologically  it 
contained  a  granular  substance  and  fascicles  of  microfilaments.  Along  its  proximal 
aspect  were  numerous  vesicles  (Fig.  2). 


FIGURE  1.  Immature  Asterias  oocyte,  5  minutes  postinsemination.  At  the  base  of  the  fertilization 
cone  is  an  incorporated  sperm  nucleus  (arrow).  G,  germinal  vesicle  containing  a  nucleolus.  x  1,000. 

FIGURE  2.  Fertilization  cone  of  an  immature  oocyte,  containing  ground  substance  and  fascicles  of 
microfilaments  (arrows),  5  minutes  postinsemination.  Along  the  base  of  the  fertilization  cone  are  aggre- 
gations of  vesicles  (V).  FM,  fertilization  membrane.  X  14,000. 

FIGURE  3.  Immature  oocyte,  30  minutes  postinsemination.  An  incorporated  sperm  nucleus  is 
depicted  at  the  arrow.  The  germinal  vesicle  (G)  is  structurally  similar  to  those  observed  in  unfertilized 
oocytes.  X  1,200. 

FIGURE  4.  Sperm  nucleus  (SN)  incorporated  into  an  immature  oocyte.  In  inseminated,  immature 
oocytes,  ooplasmic  organelles  surround  the  incorporated  sperm  nucleus  and  a  specialized  region,  lacking 
organelles  and  characteristic  of  fertilized  eggs  treated  with  1-MA,  is  not  observed.  YB,  yolk  bodies. 
XI  0,000. 


456 


F.   J.   LONGO  AND  A.   W.   SCHUETZ 


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PRONUCLEAR  DEVELOPMENT  IN  ASTERIAS  457 

Sperm  nuclei  incorporated  into  germinal  vesicle  eggs  initially  were  found  within 
the  cortex;  with  time  (i.e.,  by  30  minutes  postinsemination)  however,  they  were 
distributed  throughout  the  cytoplasm  without  any  apparent  relation  to  ooplasmic 
components  (Fig.  3).  The  cytoplasmic  area  which  surrounded  incorporated  sperm 
nuclei  was  unspecialized  in  that  it  contained  the  same  content  of  organelles  and 
inclusions  as  observed  in  other  portions  of  the  oocyte  (Fig.  4).  When  inseminated 
oocytes  were  maintained  at  20°C  with  gentle  agitation,  sperm  nuclei  remained  un- 
changed for  up  to  5  hours.  Throughout  this  period  the  sperm  nuclear  envelope 
remained  intact  and  the  condensed  sperm  chromatin  showed  no  signs  of  dispersion 
(Fig.  4).  Moreover,  the  process  of  insemination  and  the  presence  of  incorporated 
sperm  did  not  appear  to  have  any  morphological  effect  on  the  germinal  vesicle 
(Fig.  3). 

By  15  minutes  following  the  addition  of  1-MA  profound,  structural  alterations 
were  apparent  in  the  germinal  vesicle,  as  well  as  with  the  ooplasm  surrounding 
incorporated  sperm  nuclei.  The  cytoplasmic  area  surrounding  the  sperm  nucleus 
became  clear  of  organelles,  such  as  yolk  bodies  and  mitochondria,  and  within  this 
region  accumulated  endoplasmic  reticulum  and  ground  substance  (Figs.  5,  6).  This 
cytoplasmic  area  enlarged  to  greater  than  12  /j,m  in  diameter  before  morphological 
changes  were  noted  within  sperm  nuclei  (Fig.  7).  In  many  polyspermic  eggs  more 
than  one  sperm  nucleus  was  associated  with  such  a  specialized  area  (Fig.  8). 

By  30  minutes  following  the  addition  of  1-MA,  changes  in  incorporated  sperm 
nuclei  had  become  apparent.  Dilation  of  the  perinuclear  cisternae  was  pronounced, 
and  alterations  in  the  density  and  composition  of  the  condensed  chromatin  occurred. 
The  actual  disruption  or  removal  of  the  sperm  nuclear  envelope,  similar  to  that  seen 
in  other  species  (Longo,  1973),  was  not  observed,  and  how  this  membranous  struc- 
ture was  modified  to  accompany  the  expansion  of  the  paternally  derived  chromatin 
was  not  obvious  (Fig.  9). 

Chromatin  dispersion  appeared  to  differ  from  that  described  for  pronuclear  de- 
velopment in  zygotes  of  other  species  (cf.  Longo,  1973,  1981).  The  condensed  chro- 
matin gradually  transformed  from  a  dense  substance  to  a  dispersed,  filamentous 
mass  (Figs.  9-12).  These  changes  occurred  simultaneously  throughout  the  sperm 
nucleus  except  for  that  material  bordering  the  inner  margin  of  the  nuclear  envelope 
(Figs.  9,  11).  The  condensed  chromatin  lining  the  periphery  of  the  sperm  nucleus 
remained  unchanged  until  late  in  the  development  of  the  male  pronucleus  (60  to 
90  minutes  after  the  addition  of  1-MA). 


FIGURE  5.  Inseminated,  immature  oocyte,  15  minutes  after  the  addition  of  1-MA.  The  germinal 
vesicle  is  breaking  down  ("G").  An  incorporated  sperm  nucleus,  surrounded  by  a  "clear"  cytoplasmic 
area  is  shown  at  the  arrow.  Nu,  portion  of  the  disrupting  nucleolus.  XI, 000. 

FIGURE  6.  Sperm  nucleus  (SN)  incorporated  into  an  immature  oocyte  that  was  subsequently  treated 
with  1-MA  ( 15  minutes  after  the  addition  of  1-MA).  A  cytoplasmic  region,  relatively  devoid  of  organelles, 
is  associated  with  the  sperm  nucleus.  The  arrow  depicts  a  portion  of  the  sperm  nuclear  envelope  in  which 
the  perinuclear  cisterna  is  dilated.  XI  5,000. 

FIGURE  7.  Inseminated,  immature  oocyte  subsequently  treated  with  1-MA.  Sperm  nuclei  which 
are  surrounded  by  an  area  relatively  free  of  cytoplasmic  organelles  are  shown  at  the  arrows.  Sample  fixed 
30  minutes  after  the  addition  of  1-MA.  XI, 000. 

FIGURE  8.  Two  sperm  nuclei  (SN)  of  a  polyspermic,  immature  oocyte  treated  with  1-MA  for  15 
minutes.  Although  the  condensed  chromatin  does  not  show  any  recognizable  changes  when  compared 
to  oocytes  not  treated  with  1-MA,  the  perinuclear  cisternae  of  the  sperm  nuclear  envelopes  are  dilated. 
X20,000. 


458 


F.   J.   LONGO  AND  A.   W.   SCHUETZ 


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PRONUCLEAR  DEVELOPMENT  IN  ASTERIAS  459 

Changes  in  sperm  nuclear  morphology  were  not  uniform  as  there  was  consid- 
erable asynchrony  in  pronuclear  development  in  fertilized  oocytes  treated  with  1- 
MA  (Fig.  12).  This  asynchrony  appeared  to  be  a  temporal  one,  since  eventually  all 
incorporated  sperm  nuclei  developed  into  male  pronuclei.  The  relation  of  this  asyn- 
chrony to  a  specific  location  within  the  zygote,  e.g.,  the  site  of  germinal  vesicle 
breakdown  or  sperm  entry,  was  not  apparent. 

One  to  2  hours  following  the  addition  of  1-MA,  male  pronuclei  were  observed 
with  well-dispersed  chromatin  and  continuous  nuclear  envelopes  (Fig.  13).  The 
nuclear  envelope  did  not  demonstrate  the  dilations  of  the  perinuclear  cisternae 
characteristic  of  metamorphosing  incorporated  sperm  nuclei.  Internally,  clear  areas, 
surrounded  by  a  granular  nucleoplasm,  filled  the  male  pronucleus.  Nucleoli  com- 
posed of  a  dense  granular  material  also  appeared  within  developed  male  pronuclei. 
The  male  pronuclei  continued  to  enlarge  and  by  1 20  minutes  following  the  initiation 
of  pronuclear  development  measured  5  to  10  /j.m  in  diameter.  Large,  irregular  male 
pronuclei  were  also  observed  within  polyspermic  zygotes  suggesting  that  the  pro- 
nuclei  fused  with  one  another  (Fig.  14). 

Following  the  completion  of  male  pronuclear  development  the  cytoplasmic 
areas,  characteristically  associated  with  transforming  sperm  nuclei,  were  greatly  re- 
duced in  size  relative  to  the  size  of  the  male  pronucleus.  Male  pronuclei  were 
surrounded  by  cytoplasmic  areas  containing  ground  substance  and  some  endo- 
plasmic  reticulum.  This  morphology  persisted  for  approximately  2.5  hours  after  the 
addition  of  1-MA,  at  which  time  the  pronuclei  demonstrated  changes  characteristic 
of  prophase,  i.e.,  chromosome  condensation  and  nuclear  envelope  breakdown.  Con- 
comitant with  these  changes  spindles  were  formed  in  association  with  the  condensing 
paternally  derived  chromosomes;  the  numerous  mitotic  figures  that  were  produced 
were  observed  throughout  the  fertilized  egg  (Fig.  15).  Of  the  inseminated  immature 
oocytes  treated  with  1-MA  for  4  hours,  less  than  10%  cleaved  into  what  appeared 
to  be  "normal"  embryos.  Most  underwent  a  succession  of  divisions  such  that 
"morula"-like  structures,  consisting  of  blastomeres  of  different  sizes,  were  produced 
(Fig.  16). 

DISCUSSION 

The  microscopic  observations  presented  here  document  changes  induced  by  1- 
MA  treatment  on  sperm  nuclei  incorporated  into  immature  Asterias  eggs.  Mor- 
phological changes  in  the  germinal  vesicle  or  incorporated  sperm  nuclei  were  not 
apparent  for  up  to  5  h  in  the  absence  of  1-MA.  With  the  addition  of  1-MA  dramatic 


FIGURE  9.  Sperm  nuclei  (SN)  of  an  immature  oocyte  treated  with  1-MA  for  30  minutes.  The 
condensed  sperm  chromatin  is  dispersing  except  for  that  which  lines  the  nuclear  envelope.  X28.000. 

FIGURE  10.  Transforming  sperm  nuclei  (arrows)  in  an  immature  oocyte  treated  with  1-MA  for  45 
minutes.  Around  each  of  the  developing  pronuclei  is  a  specialized  cytoplasmic  region  lacking  organelles. 
XI, 300. 

FIGURE  11.  Transforming  sperm  nucleus  in  an  immature  oocyte  treated  with  1-MA  for  45  minutes. 
The  condensed  chromatin  is  dispersed  except  for  that  located  along  the  periphery  of  the  transforming 
sperm  nucleus.  X 29,000. 

FIGURE  12.  Transforming  sperm  nuclei  at  early  (small  arrows)  and  later  (large  arrows)  stages  of 
pronuclear  development.  The  earlier  stages  are  distinguished  by  dense  chromatin.  Notice  that  the  more 
developed  male  pronuclei,  i.e.,  those  with  the  more  dispersed  chromatin,  lack  the  specialized  cytoplasmic 
areas  characteristic  of  earlier  stages.  X 1 ,200. 


460 


F.   J.   LONGO  AND  A.   W.   SCHUETZ 


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FIGURE  13.  Male  pronucleus  in  a  fertilized  oocyte  treated  with  1-MA  for  60  minutes.  A  nuclear 
envelope  defines  the  border  of  the  pronucleus  which  is  surrounded  by  cytoplasm  containing  organelles. 
The  clear  areas  within  the  pronucleus  (*)  containing  some  filamentous  material  represent  areas  of  chro- 
matin  that  were  extracted  by  the  preparative  methods  employed,  x  14,000. 

FIGURE  14.  Multiple  male  pronuclei  (N)  in  a  polyspermic  oocyte  120  minutes  after  the  addition 
of  1-MA.  The  larger,  irregular  nuclear  mass  at  the  arrow  appears  to  be  derived  from  the  fusion  of  a 
number  of  pronuclei.  The  specialized  cytoplasmic  areas  associated  with  the  pronuclei  are  greatly  reduced 
in  size  relative  to  those  observed  in  earlier  specimens,  x  1,300. 

FIGURE  15.  Chromosomal  masses  (arrows)  associated  with  developing  spindles  and  derived  from 
male  pronuclei.  Specimen  prepared  150  minutes  after  the  addition  of  1-MA.  x  1,200. 

FIGURE  16.  Inseminated,  immature  oocyte  that  has  undergone  multiple  cleavages  to  form  a  "mor- 
ula"-like  structure  (incubated  with  1-MA  for  4  hours).  The  arrows  point  to  nuclei  of  the  blastomeres. 
XI, 000. 


PRONUCLEAR  DEVELOPMENT  IN  ASTERIAS 


461 


TABLE  I 

Temporal  relation  ofmeiotic  maturation,  sperm  aster  morphogenesis,  and  male  pronuclear 
development  in  fertilized  immature  Asterias  oocytes  (germinal  vesicle  intact)  subsequently 
treated  with  1-MA. 

Time  after  addition  of  1-MA  (minutes) 


Process 

0 

15 

30 

60-120 

Meiosis* 

Intact  germinal 
vesicle 

Initiation  of  germinal 
vesicle  breakdown 
(plication  of 
surface  of  germinal 
vesicle) 

Germinal  vesicle 
breakdown 
(disappearance 
of  nuclear 
envelope  and 
nucleolus) 

Development 
of  meiotic 
spindle  and 
polar  body 
formation 

Sperm  aster 

Not  present 

Present 

Increase  in  size 

Relative  reduc- 

morhogenesis 

Sperm  nucleus 
transformation 


Sperm  nucleus 
unchanged 


Sperm  nucleus 
unchanged 


Dilation  of  sperm 
nuclear 
envelope, 
chromatin 
decondensation 


tion  in  size 

Completed  male 
pronucleus 


*  Taken  from  Longo  el  al.  (1982). 


changes  were  first  noted  in  the  germinal  vesicle  and  cytoplasmic  region  associated 
with  the  sperm  nucleus.  These  were  followed  by  alterations  in  the  sperm  nucleus 
leading  to  the  formation  of  a  male  pronucleus.  Similar  results,  at  the  light  micro- 
scopic level  of  observation  with  oocytes  of  Asterina  pectinifera,  have  been  reported 
(Hirai  el  ai,  1981). 

The  structural  reorganization  of  the  cytoplasmic  area  in  association  with  the 
sperm  nucleus  was  unexpected  as  morphologically  similar  specializations,  e.g.,  asters, 
are  usually  preceded  by  the  initiation  of  pronuclear  development  in  the  zygotes  of 
other  organisms  studied  to  date  (Longo,  1973).  The  observation  that  these  regions 
developed  only  in  conjunction  with  incorporated  sperm  nuclei  suggests  that  a  sperm- 
derived  component  (e.g.,  centrioles)  is  involved  in  their  formation,  possibly  as  an 
organizing  center.  The  association  of  these  specialized  cytoplasmic  regions  with 
sperm  nuclei  and  the  fact  that  they  are  reminiscent  of  structures  earlier  microscopists 
(Wilson,  1925;  cf.  Hirai  el  al.,  1981)  referred  to  as  sperm  asters,  prompts  us  to  refer 
to  them  in  a  similar  manner. 

The  relation  of  asters  and  their  development  to  germinal  vesicle  breakdown  is 
controversial  (cf.  Masui  and  Clarke,  1979).  The  absence  of  asters  in  fertilized  im- 
mature starfish  eggs  and  their  development  in  association  with  1-MA-induced 
meiotic  maturation  as  demonstrated  herein  suggests  that  aster  formation  is  related 
to  germinal  vesicle  breakdown  (cf.  also  Franklin,  1965;  Longo,  1978)  and  is  sup- 
ported by  investigations  in  which  cellular  components,  such  as  basal  bodies,  initiate 
the  development  of  asters  when  injected  into  mature  but  not  immature  amphibian 
eggs  (Heidemann  and  Kirschner,  1975).  On  the  other  hand,  enucleation  experiments 
with  amphibian  eggs  have  suggested  that  aster  formation  is  independent  of  a  con- 
tribution of  germinal  vesicle  materials  (Katagiri,  1974;  Skoblina,  1974,  1976). 

This  and  previous  studies  implicate  the  germinal  vesicle  as  a  source  of  factors 
necessary  for  the  transformation  of  the  spermatozoon  into  a  male  pronucleus  in 


462  F.   J.   LONGO  AND  A.   W.   SCHUETZ 

starfish  (Hirai,  1976;  Hirai  et  al.,  1981;  Schuetz  and  Longo,  1981).  Similar  results 
indicating  the  control  of  nuclear  activity  via  factors  that  arise  from  or  appear  in 
concert  with  germinal  vesicle  breakdown  have  been  described  (Dettlaff  et  al.,  1964; 
Niwa  and  Chang,  1975;  Usui  and  Yanagimachi,  1976;  Longo,  1978;  Balakier  and 
Tarkowski,  1 980;  Hylander  et  al.,  1981).  The  failure  of  male  pronuclear  development 
in  enucleate  amphibian  eggs  supports  this  speculation  (Katagiri  and  Moriya,  1976; 
Skoblina,  1976).  Whether  this  requirement  is  the  result  of  specific  germinal  vesicle 
factors  or  arises  from  nucleo-cytoplasmic  interactions  following  germinal  vesicle 
breakdown  is  unclear  (Kishimoto  et  al.,  1981). 

Although  the  present  study  of  male  pronuclear  development  in  Asterias  em- 
ployed polyspermic,  immature  oocytes,  it  is  noteworthy  that  the  transformation  of 
the  sperm  nucleus  in  this  particular  system  differed  from  that  described  for  other 
species  (Longo,  1973).  Such  differences  include  the  retention  of  the  sperm  nuclear 
envelope,  the  simultaneous  dispersion  of  chromatin  throughout  the  sperm  nucleus, 
and  the  formation  of  the  male  pronuclear  envelope.  Changes  in  the  sperm  nuclear 
envelope  were  not  readily  apparent  in  fertilized,  i m mature  A sterias  eggs  treated  with 
1-MA.  This  membranous  structure  did  not  appear  to  break  down  by  a  process  of 
vesiculation  as  demonstrated  in  zygotes  of  many  species  examined  thus  far  (Longo, 
1973,  1981).  Aside  from  the  dilation  of  its  perinuclear  cisterna,  the  sperm  nuclear 
envelope  persisted  intact  throughout  pronuclear  development.  This  and  the  dramatic 
increase  in  nuclear  volume  during  pronuclear  development  raises  questions  as  to 
how  the  membrane  comprising  the  nuclear  envelope  is  augmented  to  accommodate 
the  increase  in  chromatin  dispersion.  The  dilated  perinuclear  cisternae  characteristic 
of  sperm  nuclei  within  Asterias  oocytes  treated  with  1-MA  may  be  a  manifestation 
of  this  augmentation.  In  addition,  vesicles  were  occasionally  observed  adjacent  to 
the  surface  of  the  developing  male  pronucleus  (F.  J.  Longo,  personal  observations). 
Although  we  have  not  been  able  to  document  such  an  event,  it  is  possible  that  these 
vesicles  fuse  with  and  augment  the  existing  sperm  nuclear  envelope. 

Dispersion  of  the  condensed  sperm  chromatin  in  Asterias  oocytes  differed  from 
that  described  for  sea  urchins,  where  decondensation  was  initiated  along  the  pe- 
riphery of  the  sperm  nucleus  and  progressively  appeared  more  centrad.  The  pattern 
observed  in  Asterias,  i.e.,  where  dispersion  occurred  simultaneously  throughout  the 
whole  of  the  sperm  nucleus,  is  similar  to  that  described  for  Barnea,  Callus,  and  the 
hamster  (Pasteels,  1963;  Okamura  and  Nishiyama,  1978;  Longo  and  So,  1982). 

It  has  been  suggested  that  the  asynchrony  in  pronuclear  development,  charac- 
teristic of  polyspermic  Asterias  oocytes  treated  with  1-MA,  may  be  related  to  the 
proximity  of  sperm  nuclei  with  the  germinal  vesicle  (Schuetz  and  Longo,  1981). 
This  spacial  relation  may  be  involved,  but  the  observations  made  during  the  course 
of  this  study,  where  the  extent  of  pronuclear  development  was  not  always  correlated 
with  the  site  of  germinal  vesicle  breakdown,  suggest  that  other  factors  may  have  a 
bearing  as  well.  Previous  investigations  with  mammalian  eggs  have  shown  that  the 
degree  of  polyspermy  has  a  profound  influence  on  pronuclear  development  (Hunter, 
1 967;  Hirao  and  Yanagimachi,  1979;  Witkowska,  1981).  In  these  studies  the  number 
of  sperm  developing  into  male  pronuclei  was  inversely  related  to  the  degree  of 
polyspermy;  some  sperm  nuclei  metamorphosed  into  male  pronuclei  while  the  re- 
mainder were  delayed  at  an  earlier  stage  of  pronuclear  development.  These  results 
suggest  that  in  polyspermic  eggs  competition  occurs  among  sperm  nuclei  for  ma- 
terials responsible  for  male  pronuclear  development.  That  eventually  all  sperm  nu- 
clei develop  into  male  pronuclei  in  Asterias  eggs  indicates  that  the  inhibition  of 
pronuclear  development  is  not  complete  but  rather  a  slowing  down  of  sperm  nuclear 
transformations. 


PRONUCLEAR  DEVELOPMENT  IN  ASTERIAS  463 

Samples  of  inseminated  oocytes  subsequently  treated  with  1-MA  and  examined 
just  prior  to  cleavage,  contained  large  nuclei  which  appeared  to  be  brought  about 
by  a  fusion  of  the  pronuclei.  The  presence  of  large  irregular-shaped  nuclei  as  shown 
in  Figure  14  is  suggestive  of  such  a  process;  pronuclear  fusion  has  also  been  described 
in  fertilized,  immature  Asterina  oocytes  treated  with  1-MA  (Hirai  et  ai,  1981). 
Eventually  all  of  the  nuclei  entered  mitosis  forming  what  appeared  to  be  individual 
spindles.  Similar  results  have  also  been  reported  for  the  eggs  of  other  organisms  (cf. 
Wilson,  1925;  Elinson,  1977).  Presumably  as  a  result  of  the  numerous  mitotic  ap- 
paratuses present,  the  zygote  is  induced  to  undergo  multiple  cleavages  (Rappaport, 
1971,  1975),  such  that  a  morula-like  structure  is  produced.  These  embryos  fail  to 
give  rise  to  normal  larvae;  they  eventually  degenerate  due  presumably  to  an  un- 
balanced genome. 

The  microscopic  observations  presented  here  further  illustrate  some  of  the  com- 
plex hormonal,  cytoplasmic  and  nuclear  interactions  that  occur  during  egg  matu- 
ration and  fertilization  in  Asterias  and  that  proper  synchronization  of  these  events 
is  crucial  for  normal  development.  The  temporal  relation  of  meiosis,  sperm  aster 
morphogenesis,  and  male  pronuclear  development  in  fertilized,  immature  Asterias 
forbesi  oocytes  subsequently  treated  with  1-MA  is  outlined  in  Table  I;  a  similar 
relation  has  been  described  for  Asterina  pectinifera  (Hirai  et  al.,  1981). 

ACKNOWLEDGMENTS 

Portions  of  this  investigation  were  supported  by  funds  from  the  NSF  and  the 
NIH  (HD070401-05).  Appreciation  is  expressed  to  Leslee  Miller,  Joyce  Kline,  and 
Frederick  So  for  their  assistance. 


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Reference:  Biol.  Bull.  163:  465-476.  (December  1982) 


MEIOTIC  MATURATION  AND  THE  CORTICAL  GRANULE 
REACTION  IN  STARFISH   EGGS 

FRANK  J.   LONGO,1*   FREDERICK  SO,2   AND  ALLEN   W.   SCHUETZ3 

^Department  of  Anatomy.  University  of  Iowa,  Iowa  City.  IA  52242. 

^Department  of  Population  Dynamics.  John  Hopkins  School  of  Hygiene.  Baltimore  MD  21205, 
and  the  '•2J Marine  Biological  Laboratory.  Woods  Hole,  MA  02543 


ABSTRACT 

Correlative  light  and  electron  microscopic  studies  of  immature  and  maturing 
starfish  (Asterias forbesi)  eggs  have  been  carried  out  demonstrating  ( 1 )  morphological 
alterations  attending  meiotic  maturation  induced  by  1-methyladenine  and  (2)  the 
structure  of  the  egg  cortex  and  cortical  granule  reaction.  Because  cortical  granule 
components,  are  structurally  recognizable,  their  fate  and  relation  to  the  development 
of  the  fertilization  membrane  could  be  determined.  One  and  possibly  more  of  the 
cortical  granule  components  become  an  integral  part  of  the  fertilization  membrane. 
Comparison  of  maturing  and  immature  ova  indicate  that  germinal  vesicle-contain- 
ing oocytes  (immature)  are  capable  of  undergoing  a  cortical  granule  reaction  mor- 
phologically similar  to  that  of  eggs  having  undergone  germinal  vesicle  breakdown 
(maturing). 


INTRODUCTION 

In  the  starfish,  spawning  and  oocyte  maturation  are  stimulated  by  the  ovarian 
hormone,  1-methyladenine  ( 1-MA),  which  is  synthesized  by  follicle  cells  (Kanatani 
et  #/.,  1969).  Isolated  oocytes  undergo  germinal  vesicle  breakdown,  shedding  of 
follicular  cells,  and  maturation  in  response  to  externally  applied  1-MA  at  micromolar 
concentrations.  Furthermore,  application  of  1-MA  promotes  uniform  and  synchro- 
nous maturation,  thereby  facilitating  the  study  of  oocyte  maturation,  fertilization, 
and  early  development.  Ultrastructural  investigations  of  starfish  oocytes  have  ex- 
amined oocyte-follicle  cell  relationships  and  surface  changes  stimulated  by  1-MA 
(Hirai  et  al.,  1971;  Rosenberg  et  ai,  Schroeder  et  #/.,  1979).  As  far  as  we  are  aware, 
correlative  light  and  electron  microscopic  studies  of  germinal  vesicle  breakdown  and 
meiotic  maturation  in  Asterias  oocytes  treated  with  1-MA  have  not  been  presented. 

In  addition,  although  the  fine  structure  of  the  cortex  of  fertilized  Asterias  eggs 
has  been  examined  (cf.  Monroy,  1965),  ultrastructural  analysis  of  the  cortical  granule 
reaction  in  this  organism  has  not  been  presented.  Stages  before,  during,  and  after 
the  cortical  granule  reaction  in  the  starfish,  Patina  miniata,  have  been  described 
(Holland,  1980).  In  this  study  Holland  (1980)  discussed  the  question  of  the  presence 
of  a  hyaline  layer  in  activated  starfish  oocytes  and  suggested  that  observations  made 
with  Patiria  are  representative  of  the  cortical  granule  reaction  in  other  asteroids. 
The  descriptions  of  the  cortical  granule  reaction  in  Asterias  presented  herein  have 
been  carried  out  in  light  of  Holland's  speculations. 


Received  24  May  1982;  accepted  7  September  1982. 

*  Author  to  whom  correspondence  should  be  addressed  (at  Univ.  of  Iowa). 


465 


466 


F.   J.   LONGO  ET  AL. 


Nu 


X 


ACTIVATION  OF  STARFISH   EGGS  467 

MATERIALS  AND  METHODS 

Germinal  vesicle-intact  oocytes  were  obtained  from  ripe  starfish  (Asterias forbesi) 
ovaries  which  had  been  washed  previously  in  calcium-free  sea  water  (CaFSW; 
Schuetz  and  Riggers,  1968).  Washing  in  CaFSW  inhibited  spontaneous  nuclear 
maturation  and  induced  the  detachment  of  follicle  cells  from  the  oocytes.  Ovaries 
were  minced  and  germinal  vesicle-intact  oocytes  were  separated  from  follicle  cells 
and  returned  to  artificial  sea  water  containing  the  normal  concentration  of  calcium 
(MBL  formula;  Cloud  and  Schuetz,  1973).  Germinal  vesicle  breakdown  was  induced 
by  adding  1-MA  ( 1  jug/ml;  Sigma)  to  an  oocyte  suspension,  and  samples  were  taken 
at  regular  intervals  and  fixed  for  1  hour  at  4°C  in  a  solution  of  sea  water  containing 
2%  gluteraldehyde,  0.5%  paraformaldehyde,  1%  acrolein,  1%  sodium  citrate,  and 
4.5%  sucrose.  The  samples  were  washed  overnight  in  sea  water,  incubated  in  0.5% 
OsO4  for  30  minutes,  dehydrated  in  ethanol,  and  embedded  in  Spurr  embedding 
medium.  Manipulation  of  the  specimens  during  these  procedures  has  been  described 
(Longo  and  Anderson,  1972).  Thick  sections  were  stained  with  1%  toludine  blue 
and  analyzed  with  a  Leitz  Orthoplan  microscope.  Thin  sections  were  stained  with 
uranyl  acetate  and  lead  citrate  and  examined  in  a  Philips  300  electron  microscope. 

In  order  to  examine  the  cortical  granule  reaction,  germinal  vesicle-intact  eggs, 
induced  to  mature  to  the  first  metaphase  of  meiosis  with  1  -MA,  and  ova  collected 
from  spontaneously  ovulating  females  which  had  undergone  germinal  vesicle  break- 
down were  inseminated  with  sperm  collected  from  isolated  testes.  Just  prior  to 
insemination  sperm  were  diluted  to  0.1%  (v/v)  in  sea  water.  Specimens  were  fixed 
for  1  hour  at  30-second  intervals  and  then  at  1 -minute  intervals  for  up  to  10  minutes 
post  insemination.  Further  processing  was  carried  out  as  described  above.  In  this 
report  oocytes  containing  a  germinal  vesicle  are  referred  to  as  "GV-intact"  or  "im- 
mature" ova;  oocytes  that  have  undergone  germinal  vesicle  breakdown  are  referred 
to  as  "maturing"  eggs. 

RESULTS 

Germinal  vesicle  oocytes 

The  germinal  vesicle  of  the  Asterias  oocyte  was  a  large  spheroid  body,  containing 
a  homogeneous  nucleoplasm  in  which  was  suspended  a  single  nucleolus  (Fig.  1). 
The  nucleolus  was  composed  of  a  dense,  fine-textured  material  containing  one  or 
more  areas  of  lesser  density.  The  periphery  of  the  germinal  vesicle  was  delinated  by 
a  smooth-surfaced  nuclear  envelope  (Fig.  2).  Spheroid  yolk  bodies,  containing  a 
dense  homogenous  substance,  were  found  in  close  association  with  one  another;  in 
these  instances  their  juxtaposed  surfaces  were  flattened.  Vesicles,  some  comparable 
in  size  to  yolk  bodies  but  not  nearly  as  numerous,  were  observed  within  the  cyto- 
plasm. In  addition,  smaller  vesicles,  some  with  a  filamentous  material,  others  lacking 


FIGURE  1.  Asterias  oocyte  containing  a  germinal  vesicle  (G)  and  nucleolus  (Nu)  consisting  of  two 
structural  components.  The  granularity  of  the  cytoplasm  is  due  to  yolk  bodies  and  mitochondria,  x  1 1 50. 

FIGURE  2.  Electron  micrograph  depicting  a  portion  of  the  germinal  vesicle  (G)  and  adjacent  cy- 
toplasm. The  yolk  bodies  are  frequently  found  as  aggregates  seen  at  the  arrow.  M,  mitochondria;  ER, 
endoplasmic  reticulum;  NE,  nuclear  envelope.  XI  5,000. 

FIGURE  3.  Portion  of  a  germinal  vesicle  initiating  the  resumption  of  meiosis  (i.e.,  germinal  vesicle 
breakdown),  30  minutes  after  exposure  to  1-MA.  The  vesicles  depicted  by  the  arrows  are  presumably 
products  of  the  vesiculation  of  the  nuclear  envelope.  Nu,  disrupting  nucleolus.  x  10,000. 


468 


F.  J.   LONGO  ET  AL. 


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ACTIVATION  OF  STARFISH   EGGS  469 

a  substructure,  were  also  present  (Fig.  2).  Small  cisternae  of  endoplasmic  reticulum, 
mostly  of  the  smooth  variety,  as  well  as  mitochondra  were  distributed  throughout 
the  cytoplasm.  Golgi  complexes  were  not  prominent. 

Germinal  vesicle  breakdown 

Breakdown  of  the  germinal  vesicle  in  oocytes  matured  naturally  or  with  exo- 
geneous  1-MA  was  morphologically  similar;  the  observations  provided  below  are 
taken  from  studies  where  maturation  was  initiated  by  exogenous  1-MA.  Further- 
more, the  application  of  1-MA  to  immature  oocytes  allowed  for  a  precise  timing 
of  meiotic  maturation.  Hence,  the  times  referred  to  herein  are  based  on  counts  of 
eggs  where  greater  than  50%  demonstrated  a  given  stage  of  development  (N  >  100) 
and  where  the  moment  of  addition  of  1-MA  was  time-zero. 

One  of  the  earliest  signs  of  germinal  vesicle  breakdown  was  the  modification  of 
the  periphery  of  the  germinal  vesicle;  i.e.,  its  surface  became  convoluted  by  15 
minutes  after  the  addition  of  1-MA,  and  this  was  followed  by  the  disruption  of  the 
nuclear  envelope  (Figs.  3,  4).  The  nuclear  envelope  vesicutated,  such  that  numerous 
vesicles  were  found  along  the  interface  of  cytoplasm  and  the  nucleoplasm  (Fig.  3). 
Concomitantly,  the  nucleolus  assumed  a  highly  irregular  profile  and  dispersed  (Fig. 
4).  Continued  meiotic  maturation  led  to  a  considerable  reduction  in  the  volume 
formerly  occupied  by  the  germinal  vesicle  (Figs.  5-7).  At  30  minutes  after  the  ad- 
dition of  1-MA  the  nuclear  envelope  and  much  of  the  nucleolus  had  disappeared. 
By  40  minutes  after  the  addition  of  1-MA  the  condensing  chromosomes  were  ap- 
parent as  "clear"  areas  rather  than  the  usual  opaque  structures  obtained  in  fixed 
preparations  of  other  cell  types  (Fig.  6).  Evidently,  the  preparative  methods  employed 
in  this  study  removed  portions  of  the  chromosomes. 

Within  60  minutes  following  the  application  of  1-MA,  the  chromosomes  were 
observed  associated  with  the  forming  meiotic  spindle  which  was  usually  located  in 
the  central  portion  of  the  egg  (Figs.  6,  7).  Relative  to  the  size  of  the  egg  the  meiotic 
apparatus  was  small,  measuring  about  10  ^m  in  length.  It  lacked  prominent  asters 
and  appeared  "barrel-shaped"  when  sectioned  longitudinally.  The  spindle  migrated 
to  the  animal  pole  of  the  egg  and  underwent  its  meiotic  divisions  (Figs.  8,  9). 

The  second  polar  body  of  a  fertilized  starfish  egg  is  shown  in  Figure  10,  120 
minutes  after  addition  of  1-MA.  The  chromosomes  taken  into  the  second  polar 


FIGURES  4-6.  Asterias  oocytes  in  successive  stages  of  meiotic  maturation.  Eggs  fixed  at  15  (Fig. 
4),  30  (Fig.  5),  and  40  minutes  (Fig.  6)  after  the  addition  of  1-MA.  xl  150.  Figure  4  depicts  the  development 
of  plications  along  the  periphery  of  the  germinal  vesicle  (arrows),  the  disruption  of  the  nucleolus  (Nu), 
and  the  development  of  a  "clearing"  (C)  of  organelles  along  the  periphery  of  the  germinal  vesicle.  This 
is  followed  by  the  disappearance  of  the  nuclear  envelope  (Fig.  5)  and  the  condensation  of  the  meiotic 
chromosomes  (Ch),  which  are  the  lightly  stained  structures  shown  in  the  center  of  the  egg  in  Figure  6. 
The  structures  depicted  by  arrows  in  Figure  5  are  remnants  of  the  nucleolus. 

FIGURES  7  AND  8.  Condensed  chromosomes  (arrows)  organized  on  the  meiotic  spindle  (MS)  which 
is  formed  in  the  center  of  the  egg  (Fig.  7)  and  then  moves  to  the  cortex  (Fig.  8).  Specimens  prepared  at 
60  and  70  minutes  after  addition  of  1-MA,  respectively. 

FIGURE  9.  Asterias  egg  having  completed  the  formation  of  the  first  polar  body  (1PB);  specimen 
fertilized  after  germinal  vesicle  breakdown.  The  chromosomes  remaining  within  the  egg  are  shown  at  the 
arrow,  prior  to  their  organization  on  the  second  meiotic  spindle.  XI 150. 

FIGURE  10.  Second  polar  body  of  a  fertilized  Asterias  egg  located  within  the  perivitelline  space 
(PVS)  and  containing  a  nucleus  (N)  and  centriole  (C).  The  areas  within  the  nucleus  indicated  (*)  represent 
chromatin  which  is  dissolved  by  the  preparative  methods  employed,  x  36,000. 


470 


F.   J.   LONGO  ET  AL. 


MV 


ACTIVATION  OF  STARFISH   EGGS  471 

body  comprised  a  miniature  nucleus.  In  addition  to  mitochondria  and  some  vesic- 
ular structures,  at  least  one  centriole  was  observed  within  the  second  polar  body. 

Cortical  granule  reaction 

The  cortex  of  immature  and  maturing  (from  both  1-MA  treated  specimens  and 
spontaneously  ovulating  females)  oocytes  appeared  morphologically  similar  to  one 
another.  The  plasma  membrane  was  projected  into  numerous  microvilli  that  were 
arranged  in  a  hexagonal  pattern  and  were  covered  by  a  prominent  vitelline  layer, 
composed  of  a  filamentous  material  (Figs.  11-13).  Although  in  some  specimens  the 
vitelline  layer  was  separated  slightly  from  the  surface  of  the  egg  and  only  covered 
the  tips  of  the  microvilli  (Fig.  12),  it  was  morphologically  similar  to  those  that 
surrounded  the  microvilli  (Figs.  11,  13).  The  separation  of  the  vitelline  layer  was 
a  random  occurrence,  seemingly  unrelated  to  1-MA  treatment;  its  basis  was  not 
established.  A  monolayer  of  ellipsoid  cortical  granules  was  located  within  the  cortex 
(Figs.  11,  12).  The  long  axis  of  the  granules  was  positioned  at  a  right  angle  to  the 
surface  of  the  egg  and  measured  2  to  2.5  ^m  in  length  (Fig.  1 1).  Structurally  the 
granules  usually  contained  three  components  that  were  resolved  at  high  magnifi- 
cation (Fig.  14).  The  first  was  a  spheroid  mass  of  dense  material,  having  a  fine- 
textured  appearance,  which  was  positioned  in  the  distal  and/or  proximal  portions 
of  the  granule.  Second,  was  a  fine  granular  material  of  lesser  electron  opacity  that 
often  surrounded  the  first  and  filled  much  of  the  remainder  of  the  granule.  The  third 
component  was  dense,  relatively  sparse  in  comparison  to  the  other  two  components, 
and  usually  confined  to  the  lateral  aspect  of  the  granule.  In  addition  to  the  cortical 
granules  some  vesicular  elements  and  mitochondria  were  present  in  the  cortex  of 
maturing  and  immature  oocytes  (Figs.  11,  14). 

Insemination  initiated  the  cortical  granule  reaction  which  was  morphologically 
the  same  in  immature  and  maturing  eggs  and  comparable  to  that  previously  de- 
scribed for  sea  urchins  (Anderson,  1968;  Millonig,  1969).  Consequently,  only  mi- 
crographs of  the  cortical  granule  reaction  in  maturing  eggs  are  presented.  Because 
of  the  three  morphologically  distinct  components  of  the  starfish  cortical  granule, 
their  fate  and  relation  to  one  another  with  respect  to  the  formation  of  the  fertilization 
membrane  and  organization  within  the  perivitelline  space  could  be  followed.  A 
dehiscing  cortical  granule  is  shown  in  Figure  15.  Soon  after  fusion  of  the  cortical 
granule  membrane  with  the  plasma  membrane  all  three  components  of  the  cortical 


FIGURE  1  1.  Cortex  of  an  immature  Asterias  oocyte  containing  cortical  granules  (CG).  The  plasma 
membrane  is  covered  by  a  vitelline  layer  (VL).  Three  structural  components  (1,2,  and  3;  see  text  for 
explanation)  may  be  seen  in  some  cortical  granules,  x  27,000. 

FIGURE  12.  Portion  of  the  cortex  of  an  unfertilized  maturing  oocyte  in  which  the  vitelline  layer 
(VL)  has  separated  from  the  plasma  membrane.  Under  these  conditions  the  vitelline  layer  shows  no 
changes  in  internal  structure.  MV,  microvilli;  CG,  cortical  granules,  x  30,000. 

FIGURE  13.  Tangential  section  of  the  surface  of  a  maturing  Asterias  egg  demonstrating  the  orga- 
nization of  microvilli  (MV).  The  filamentous  material  eminating  from  the  microvilli  represents  a  part 
of  the  vitelline  layer.  X49,000. 

FIGURE  14.  Cortical  granule  of  a  maturing  Asterias  oocyte  containing  three  structural  components 
(1,2,  and  3;  see  text).  A  portion  of  the  vitelline  layer  (VL)  is  depicted.  x33,000. 

FIGURE  15.  Dehiscing  cortical  granule  of  an  inseminated,  maturing  oocyte.  Initially,  the  more 
electron  translucent  component  (2)  is  closely  associated  with  the  vitelline  layer  (VL)  which  is  separating 
from  the  surface  of  the  egg.  Other  components,  which  are  still  confined  to  the  cup-like  structure  of  the 
dehiscing  cortical  granule  (1  and  3),  are  beginning  to  disperse.  X52,000. 


472 


F.  J.   LONGO  ET  AL. 


FM 


PVS 


I 


FM 


>: 


PVS 


I 


I 


-       - 

• 

- 


ACTIVATION  OF  STARFISH   EGGS  473 

granule  appeared  to  swell.  Initially  the  second  component  became  associated  with 
the  vitelline  layer.  Later,  however,  much  of  this  material  appeared  to  disperse  and 
fill  the  perivitelline  space  (Figs.  16,  17).  The  first  component  became  associated  with 
the  vitelline  layer;  this  material  eventually  coated  the  entire  inner  margin  of  the 
developing  fertilization  membrane  and  was  seen  as  a  dense  layer  (Figs.  16,  17).  The 
fate  of  the  third  component  was  unclear;  it  appeared  to  form  plate-like  structures 
that  were  distributed  throughout  the  perivitelline  space  (Figs.  16,  17). 

Following  the  release  of  the  cortical  granule  contents  a  well-defined  fertilization 
membrane  was  formed  in  both  immature  and  maturing  ova  (Fig.  18).  Morpholog- 
ically, it  consisted  of:  (1)  an  outer  laminated  region  apparently  derived  from  the 
vitelline  layer  itself,  with  a  possible  contribution  from  the  second  component  of  the 
cortical  granule,  and  (2)  an  electron  opaque  region  along  the  innermost  portion  of 
Jhe  fertilization  membrane  consisting  of  material  derived  from  the  first  component 
of  the  cortical  granule  (Fig.  1 8).  The  perivitelline  space  of  both  inseminated  im- 
mature and  maturing  eggs  was  relatively  large  and  measured  up  to  12  yum  in  width 
(Fig.  19);  it  was  filled  primarily  with  an  electron  translucent  substance  in  which 
were  found  some  dense  structures  apparently  derived  from  the  cortical  granules. 

DISCUSSION 

The  observations  of  this  study  demonstrate:  ( 1 )  morphological  alterations  and 
their  chronology  in  Asterias  eggs,  induced  by  1-MA,  leading  to  the  development  of 
the  second  polar  body,  (2)  the  structure  of  the  Asterias  egg  cortex  and  cortical  granule 
reaction,  and  (3)  that  germinal  vesicle-containing  oocytes  of  Asterias  are  capable 
of  undergoing  a  cortical  granule  reaction  morphologically  similar  to  that  of  eggs 
having  undergone  germinal  vesicle  breakdown. 

Germinal  vesicle  breakdown  and  meiotic  maturation 

The  germinal  vesicle  of  Asterias  oocytes  is  morphologically  comparable  to  those 
observed  in  eggs  of  other  organisms  (Kessel,  1968;  Millonig  et  #/.,  1968;  Longo  and 
Anderson,  1970).  That  1-MA  had  an  effect  on  meiotic  maturation  was  first  indicated 
by  the  undulation  of  the  nuclear  envelope  of  the  germinal  vesicle  and  a  disruption 
of  the  nucleolus.  These  changes  are  characteristic  of  germinal  vesicle  breakdown  as 


FIGURE  16.  Cortex  of  a  maturing  oocyte,  in  which  the  contents  of  the  cortical  granules  fill  the 
perivitelline  space.  The  cortical  granule  component  designated  2  is  dispersed  within  the  perivitelline  space 
and  may  have  become  integrated  into  the  developing  fertilization  membrane  (FM).  Component  1  is  seen 
as  an  electron  dense  aggregate  closely  associated  with  the  inner  margin  of  the  developing  fertilization 
membrane.  Electron  dense  component  3  is  distributed  within  the  perivitelline  space.  X49.000. 

FIGURE  17.  Perivitelline  space  (PVS)  of  a  fertilized,  mature  oocyte  in  which  one  of  the  structural 
components  (1)  of  the  cortical  granules  has  lined  the  inner  surface  of  the  developing  fertilization  mem- 
brane. The  material  distributed  throughout  the  perivitelline  space  may  be  derived  from  component  2. 
The  dense  material  (3)  may  be  derived  from  the  third  component  of  the  cortical  granules.  X44,000. 

FIGURE  18.  Structural  organization  of  the  fertilization  membrane  (FM)  of  an  inseminated,  im- 
mature oocyte.  The  fertilization  membrane  has  collapsed  and,  consequently,  is  located  in  close  proximity 
to  the  egg  surface.  The  outermost  aspect  of  the  fertilization  membrane  (*)  consists  of  laminated  regions 
(arrows).  The  innermost  aspect  of  the  fertilization  membrane  is  lined  by  electron  dense  material  derived 
from  component  1  of  the  cortical  granules.  The  dense  material  located  within  the  perivitelline  space 
(PVS)  may  be  derived  from  component  3  of  the  cortical  granules.  X28.000. 

FIGURE  19.  Immature  ooctye  2  hours  after  insemination  possessing  a  fertilization  membrane  (FM) 
and  prominent  perivitelline  space  (PVS).  G,  germinal  vesicle.  XI 300. 


474  F.   J.   LONGO  ET  AL. 

observed  in  oocytes  of  other  organisms  where  meiotic  maturation  is  induced  by 
other  means  (Merchant  and  Chang,  1971;  Calarco  et  al.,  1972;  cf.  Longo,  1973; 
Sorenson,  1973).  The  significance  of  the  tortuous  outline  developed  by  germinal 
vesicles  induced  to  break  down  has  not  been  established.  Similar  distortions  in 
nuclear  structure  observed  in  other  cells  may  be  due  to  fluxes  of  materials  into  and 
out  of  the  nucleus  (Monroy,  1 965).  Changes  in  the  germinal  vesicle  of  starfish  oocytes 
induced  by  1-MA  are  believed  to  be  brought  about  by  the  production  of  maturation 
promotion  factor  which  is  also  responsible  for  subsequent  maturation  events  (Kish- 
imoto  et  al.,  1981). 

With  the  exception  of  the  development  of  asters,  formation  of  the  meiotic  ap- 
paratus in  Aster ias  is  similar  to  that  described  for  Spisula  and  Tubifex  (Longo  and 
Anderson,  1969,  1970;  Shimizu,  198 la,  b).  The  meiotic  spindle  was  formed  in  the 
central  portion  of  the  oocyte  and  then  moved  to  the  cortex.  The  meiotic  spindle  of 
Asterias  was  structurally  similar  to  that  observed  in  mouse  eggs  in  that  it  lacked 
well-developed  asters  and  was  barrel-shaped  (Szollosi  et  al.,  1972).  Due  to  the  rel- 
atively large  size  of  oocytes  we  were  unable  to  verify  the  appearance  and  number 
of  centrioles  in  the  meiotic  spindles  of  Asterias;  however,  the  presence  of  at  least 
one  centriole  in  the  second  polar  body  indicates  that  these  organelles  are  probably 
an  integral  part  of  the  meiotic  apparatus.  Thus,  the  situation  differs  from  that  ob- 
served in  mammals.  Characteristically,  the  meiotic  spindle  of  mammalian  oocytes 
lacks  centrioles  (Szollosi,  1972;  Szollosi  et  al.,  1972). 

Cortical  granule  reaction 

There  has  existed  in  the  literature  a  question  as  to  whether  or  not  immature 
starfish  oocytes  are  capable  of  a  cortical  granule  reaction  and  the  formation  of  a 
fertilization  membrane  (cf.  Masui  and  Clarke,  1979).  It  has  been  generally  believed 
that  germinal  vesicle  breakdown  was  necessary  before  the  starfish  egg  was  capable 
of  a  cortical  reaction  (Hirai  et  al..  1971;  Hirai,  1976).  However,  immature  eggs 
incubated  in  calcium-free  sea  water  were  able  to  inseminate  and  undergo  a  cortical 
granule  reaction  (Cayer  et  al.,  1975;  Schuetz,  1975).  The  results  presented  herein 
support  and  amplify  these  observations  at  the  ultrastructural  level  of  observation 
and  indicate  that  the  cortical  granule  reaction  in  Asterias  ova,  with  or  without 
germinal  vesicles,  is  structurally  similar. 

The  cortical  granule  reaction  in  Asterias  is  morphologically  similar  to  that  de- 
scribed by  Holland  (1980)  for  Patiria  miniata.  Because  of  structurally  recognizable 
cortical  granule  components,  their  fate  and  relation  to  development  of  the  fertil- 
ization membrane  can  be  traced.  The  present  study  shows  that  the  dense  component 
of  the  cortical  granules  coats  the  inner  margin  of  the  vitelline  layer  and  becomes 
an  integral  part  of  the  fertilization  membrane.  A  similar  process  has  also  been 
described  for  sea  urchins  and  Patiria  (Anderson,  1968;  cf.  Ito,  1969;  Inoue  and 
Hardy,  1971;  Holland,  1980). 

Investigators  working  with  the  eggs  of  different  organisms  have  shown  that  cor- 
tical granule  components  become  a  part  of  the  vitelline  layer  and  their  interaction 
is  related  to  characteristics  the  fertilization  membrane  acquires  with  its  development, 
e.g.,  hardening  (Endo,  1961;  Wolpert  and  Mercer,  1961;  Bryan,  1970;  Grey  et  al., 
1974;  Chandler  and  Heuser,  1980;  cf.  Shapiro  and  Eddy,  1980;  Schuel  et  al.,  1982). 
A  similar  interaction  may  also  exist  in  Asterias.  That  the  vitelline  layer  of  Asterias 
showed  no  change  in  structure  when  separated  from  the  surface  of  eggs  not  having 
undergone  a  cortical  reaction  suggests  that  cortical  granule  material  is  necessary  for 
the  progressive  structuralization  of  the  fertilization  membrane. 


ACTIVATION  OF  STARFISH   EGGS  475 

Despite  the  release  of  the  entire  population  of  cortical  granules  and  evidence 
from  other  echinoderms  demonstrating  that  components  of  the  hyaline  layer  are 
derived  from  cortical  granules  (Kane,  1970;  Stephens  and  Kane,  1970;  cf.  Schuel, 
1978),  a  well-defined  hyaline  layer  was  not  obvious  in  fertilized  eggs  of  Asterias. 
Although  some  of  the  cortical  granule  material  is  incorporated  into  the  fertilization 
membrane,  the  fate  of  the  remainder  is  in  question.  Some  material  is  seen  within 
the  perivitelline  space.  However,  it  is  much  too  sparse  to  form  a  prominent  layer 
as  found  in  many  sea  urchins.  One  reason  for  the  absence  of  a  layer  may  be  due 
to  the  relatively  larger  perivitelline  space,  characteristic  of  fertilized  Asterias  eggs. 
The  cortical  granule  contents  may  fill  this  space,  resulting  in  a  relatively  diffuse 
distribution.  Holland  (1980,  1981)  has  questioned  the  presence  of  a  hyaline  layer 
in  starfish  as  found  in  echinoids.  As  indicated  by  Hall  and  Vacquier  (1982),  par- 
ticipation by  the  hyaline  layer  does  not  appear  to  be  greatly  relevant  to  echinoderm 
morphogenesis,  as  this  structure  is  seemingly  found  in  only  echinoids  and  ophiuroids 
(cf.  also  Holland,  1981).  In  starfish,  the  interaction  of  blastomeres  alone  without  the 
aid  of  an  extracellular  layer  seems  to  be  sufficient  for  blastula  formation  (Dan- 
Sohkawa,  1976;  Dan-Sohkawa  and  Fujisawa,  1980). 

ACKNOWLEDGMENTS 

Portions  of  this  investigation  were  supported  by  funds  from  the  NSF  and  the 
NIH  (HD070401-05).  Appreciation  is  expressed  to  Ms.  Julie  Anolik  for  her  as- 
sistance. 

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Reference:  Biol.  Bull.  163:  477-491.  (December  1982) 


DISTRIBUTION  AND  ECOLOGY  OF  MYSIDS  IN 
CAPE  COD  BAY,   MASSACHUSETTS 

DON   MAURER   AND   ROLAND  L.   WIGLEY1 

Southern  California  Ocean  Studies  Consortium,  California  State  University,  Long  Beach,  CA  90840 

ABSTRACT 

Seven  species  of  mysids  (Neomysis  americana,  Erythrops  erythropthalma,  Mysis 
stenolepis,  Mysis  mixta,  Heteromysis  formosa,  Praunus  flexuosus,  and  Meterythrops 
robusta)  were  collected  from  Cape  Cod  Bay,  Massachusetts.  The  general  ecology  of 
the  first  four  species  is  described  in  terms  of  several  classificatory  schemes  proposed 
for  worldwide  mysid  distributions. 

Organismal  relationships  to  geographic,  seasonal,  bathymetric,  bottom  water 
temperature,  and  sediment  characteristics  are  examined.  Four  species  occurred  year- 
round  with  the  following  seasonal  peaks  in  abundance:  N.  americana  (February, 
April,  December),  E.  erythropthalma  (January,  March,  December),  M.  mixta 
(March  and  July),  M.  stenolepis  (January  and  August).  Based  on  bathymetric  and 
sedimentary  relationships  the  species  tend  to  occur  in  pairs.  Neomysis  americana 
and  M.  stenolepis  were  primarily  collected  in  shallow  water  (10-29  m)  and  from 
sand  and  clayey-silt.  Erythrops  erythropthalma  and  M.  mixta  occurred  in  deeper 
water  (20-39  m)  and  on  clayey-silt  and  silt.  In  addition  to  seasonal  effects,  evidence 
indicates  that  interactions  among  depth,  bottom  water  temperature,  and  sediment 
type  strongly  influenced  the  spatial  zonation  of  Cape  Cod  mysids.  The  distribution 
and  ecology  of  the  four  mysids  generally  conformed  to  worldwide  classification 
schemes. 

INTRODUCTION 

The  Cape  Cod  Bay,  Massachusetts  biotic  census  was  conducted  to  provide  data 
on  species  composition,  abundance,  diversity,  and  trophic  groupings  of  marine 
benthic  organisms  in  regard  to  biotic  and  abiotic  factors,  and  to  provide  a  base  for 
systematic  and  ecologic  investigations  of  the  Cape  Cod  Bay  ecosystem  and  for  as- 
sessment of  change  brought  about  by  human  activities  (Carriker,  1972).  The  present 
account  focuses  on  the  mysidaceans  from  the  biotic  census. 

Since  mysidaceans  form  a  conspicuous  component  of  macrozooplankton  in 
freshwater  and  oceanic  environments  and  can  form  an  important  resource  in  food 
web  dynamics,  they  have  been  extensively  studied  (Gordan,  1957).  Research  along 
the  northeast  coast  of  North  America  reflects  this  worldwide  interest  (Verrill  et  al., 
1873;Rathbun,  1905;  Bigelow  and  Sears,  1939;  Bousfield,  1956,  1961;  Brunei,  1960; 
Wigley,  1963;  Haefner,  1968).  Although  mysids  are  commonly  considered  to  be 
planktonic,  studies  with  a  variety  of  bottom  collectors  have  shown  that  some  species 
are  benthic  or  spend  some  portion  of  their  life  on  the  bottom  (Clutter,  1 967;  Murano, 
1970a,  b).  Based  on  3  X  106  specimens  collected  from  1953  to  1969  from  the  con- 
tinental shelf  and  slope  between  Canada  and  southern  Florida,  bathymetry,  bottom 

Received  7  June  1982;  accepted  7  September  1982. 

1  Present  address:  35  Wilson  Road,  Woods  Hole,  MA  02543. 

Contribution  Number  7  from  Southern  California  Ocean  Studies  Consortium. 

477 


478 


D.   MAURER   AND  R.   L.   WIGLEY 


sediment,  and  generation  type  were  recognized  as  important  features  influencing 
mysid  distribution  (Wigley  and  Burns,  1971).  In  a  comprehensive  review  Muachline 
(1980)  proposed  several  classifications  to  describe  worldwide  mysid  distributions 
based  on  these  features.  This  research  examines  whether  mysidaceans  collected  by 
the  Cape  Cod  Bay  biotic  census  were  responding  to  the  same  features  underlying 
Mauchline's  (1980)  classifications  for  worldwide  distributions. 

Cape  Cod  Bay  is  described  elsewhere  (Young  and  Rhoads,  1971).  It  encompasses 
1600  km2,  is  circular,  and  opens  northward  to  Massachusetts  Bay  (Figure  1).  Mean 
tidal  range  at  Plymouth,  Massachusetts  is  2.9  m.  Average  annual  extremes  of  surface 
temperature  (—0. 1  and  1 9.9°C)  and  salinity  (31.0  and  33.2%o)  are  similar  to  bottom 
temperature  (-0.1  and  17.7°C)  and  bottom  salinity  (31.2  and  32.3%o).  Bottom 
temperatures  ranged  from  -1.5  to  23.5°C.  Highest  and  lowest  values  of  both  hy- 
drographic  features  are  normally  associated  with  surface  waters.  A  summer  ther- 
mocline  appears  in  April  and  disappears  in  October.  Reverse  thermoclines  may 
occur  at  15  to  25  m  during  mid-winter  when  bottom  water  may  be  1  to  2.5°C 
warmer  than  surface  water.  Sediments  consist  of  a  mixture  of  clayey-silt,  silt,  sand, 
and  gravel.  Sand  and  silt  each  comprise  approximately  40-45%  of  the  bay  sediments, 
and  gravel  comprises  the  smallest  component  of  sediment  (Young  and 
Rhoads,  1971). 

MATERIALS  AND  METHODS 

The  methods  used  to  collect  and  process  the  samples  are  described  in  detail  by 
Young  el  al.  (1971).  Since  the  goals  of  the  study  were  to  maximize  the  number  of 


CAPE    COD 


FIGURE  1 .    Location  of  station  quadrats,  Cape  Cod  Bay,  Massachusetts. 


MYSIDS  OF  CAPE  COD  BAY  479 

different  locations  sampled,  no  repetitive  sampling  over  time  was  conducted.  Cape 
Cod  Bay  was  divided  into  one  square  mile  quadrats  (Figure  1).  Sampling  was  con- 
ducted from  1966  to  1969,  yielding  samples  from  each  month  of  the  year.  Although 
the  sampling  effort  was  evenly  distributed  over  the  bay,  based  on  sediment  distri- 
bution approximately  36.6  percent  of  the  samples  were  taken  from  mud  >40  (where 
0  =  median  sediment  particle  size;  0.062  mm),  42.5  percent  from  very  fine  to  coarse 
sand  1-40  (0.062-0.50  mm),  and  20.9  percent  from  coarse  sand  to  gravel.  Thus 
there  is  bias  towards  samples  from  coarser  grained  sediment.  It  should  be  emphasized 
that  sampling  was  not  synoptic  and  that  seasonal  patterns  are  based  on  a  composite 
of  samples  collected  over  several  years.  Quadrats  were  sampled  randomly  over  sed- 
iment type  and  depth  range. 

Quantitative  samples  were  taken  from  the  center  and  four  corners  of  each  com- 
plete quadrat  by  a  Smith-Mclntyre  grab  (0. 1  m2).  Dredge  hauls  were  obtained  from 
three  of  the  corners  by  towing  to  the  center  of  each  quadrat.  The  dredge  types 
included  an  epibenthic  sled,  a  modified  commercial  clam  dredge,  and  a  naturalist 
dredge. 

Quantitative  samples  were  washed  immediately  by  elutriation  with  sea  water 
into  1.0  and  0.5  mm  screens,  and  dredge  hauls  were  washed  through  the  former. 
The  washed  residue  on  each  screen  was  placed  for  5-10  minutes  in  a  0. 15%  solution 
of  propylene  phenoxytol  in  sea  water.  Specimens  were  preserved  in  a  10%  solution 
of  formalin  in  sea  water  for  48-72  hours,  rinsed  with  tap  water  for  several  minutes, 
and  transferred  to  85%  ethyl  alcohol  for  final  storage.  Preserved  samples  of  mysids 
were  sorted  according  to  species,  sex,  and  life  stage  and  counted  under  microscopes. 

Four  hundred  and  sixty  grab  samples  and  260  dredge  hauls  were  collected.  At 
the  center  of  each  quadrat,  surface  and  bottom  temperature  and  salinity  were  mea- 
sured. Sediment  cores  for  analysis  of  particle  size  were  taken  from  each  Smith- 
Mclntyre  sample  and  frozen  until  analyzed.  A  total  of  320  sediment  samples  were 
analyzed.  Textural  analysis  was  done  by  dry  sieving  the  sand  fractions  through  an 
Udden-Wentworth  sieve  series  on  a  RoTap  shaker  following  initial  dispersion  with 
sodium  metaphosphate.  The  silt  and  clay  fractions  were  determined  by  pipette  anal- 
ysis. For  purposes  of  this  presentation  gravel  is  defined  as  >  -1.00,  sand  -1.0  to 
4.00,  silt  4.0  to  5.00,  and  clayey-silt  >5.00. 

The  number,  sex,  and  life  stage  (adult,  immature,  ovigerous,  larvigerous)  of 
individuals  per  each  species  of  mysid  were  tabulated.  The  density  (grab),  relative 
abundance  (dredge),  and  frequency  (percent  of  occurrence)  in  grab  and  dredge  sam- 
ples were  compared  to  environmental  factors  with  correlation  coefficients  (R).  Den- 
sity was  transformed  by  loge  (N  +  1 )  prior  to  correlation.  Analysis  of  covariance  was 
performed  on  monthly  density  counts  using  biomedical  computer  programs  from 
the  University  of  California,  Los  Angeles.  The  program  produced  an  analysis  of 
variance  for  adjusted  group  means  and  a  /-test  matrix  for  adjusted  group  means. 
This  procedure  was  used  because  of  unequal  data  sets  and  because  it  tests  whether 
the  means  of  the  dependent  variable  are  significantly  different  among  groups  and 
whether  the  difference  is  due  to  differences  in  the  independent  variable  among  the 
groups  (Snedecor  and  Cochran,  1967;  Sokal  and  Rohlf,  1969). 


RESULTS 


General  occurrence 


Neomysis  americana  (Smith)  was  collected  most  frequently  followed  in  descend- 
ing frequency  by  Erythrops  erythropthalma  (Goes),  Mysis  mixta  Lillgeborg,  and 
Mysis  stenolepis  Smith.  Neomysis  americana  occurred  throughout  the  bay  except 


480  D.    MAURER   AND   R.    L.   WIGLEY 

the  north  central  portion  whereas  E.  erythropthalma  and  M.  mixta  occurred  ev- 
erywhere except  the  southern  and  southeastern  portion.  My  sis  stcnolepis  occurred 
mainly  in  the  southern  half  of  the  bay  with  a  few  occurrences  in  the  northern  half. 
Several  specimens  of  Heterorn ysis  formosa  (Smith)  and  Praunus flexuosus  (Miiller) 
and  a  damaged  specimen  questionably  assigned  to  Meterythrops  robusta  (Smith) 
were  also  collected. 

Seasonal  distribution 

Neomysis  americana  was  collected  every  month  with  abundance  peaks  in  Feb- 
ruary, April,  and  December  (Table  I).  Based  on  Analysis  of  Variance  (ANOVA,  F 
=  2.48,  D.F.  66,360)  the  effect  of  month  of  collection  was  statistically  significant 
(a  =  0.01).  Examination  of  the  Mest  matrix  for  adjusted  group  means  of  grab  sam- 
ples indicated  that  catches  from  February,  April,  and  December  were  significantly 
different  (a  =  0.05)  from  those  in  other  months.  Patterns  based  on  dredge  data 
showed  high  relative  abundance  during  the  same  three  months. 

The  overall  sex  ratio  of  adults  was  0.51  males  to  1  female  (grab)  and  0.82  males 
to  1  female  (dredge).  Dredge  hauls  yielded  ovigerous  stages  in  October  and  larvi- 
gerous  stages  from  April  through  October. 

Erythrops  erythropthalma  was  collected  every  month,  and  number  collected 
peaked  in  March  and  December  (Table  I).  Based  on  ANOVA  (F  1.6,  D.F. 
66,  360),  the  effect  of  month  was  statistically  significant  (a  =  0.01 ).  The  Mest  matrix 
for  grab  data  indicated  that  January  and  December  were  significantly  different  (a 
-  0.05).  Trends  depicted  by  dredge  data  indicated  relatively  large  numbers  January 
through  April  (Table  I). 

Density  of  E.  erythropthalma  decreased  throughout  spring  and  summer  (May- 
August).  The  overall  sex  ratio  of  adults  was  0.32  males  to  1  female  (grab)  and  0.53 
males  to  1  female  (dredge).  Dominance  in  sex  ratio  of  E.  erythropthalma  changed 
more  frequently  throughout  the  year  than  did  that  of  N.  americana.  June  and 
November  were  the  only  months  when  immature  forms  were  not  collected  by  dredge. 
Dredge  collections  produced  ovigerous  stages  in  May  and  July  (Table  I).  Larvigerous 
stages  were  collected  in  January,  May-September,  and  December. 

Mysis  mixta  was  collected  every  month  but  October  (Table  I).  Based  on  ANOVA 
(F  =  2.7,  D.F.  66,360),  the  effect  of  month  of  sampling  was  significant  (a  ==  0.01). 
the  Mest  matrix  for  grab  data  indicated  that  the  majority  of  monthly  samples  were 
significantly  different  (a.  --  0.05)  from  one  another.  Dredge  data  indicated  a  peak 
in  March  followed  by  a  rapid  decline  and  gradual  increase  through  July  (Table  I). 
Immature  forms  occurred  from  April  to  July.  Ovigerous  forms  were  collected  in 
January  and  December,  and  larvigerous  stages  were  taken  from  January  to  April. 
The  sex  ratio  of  adults  was  0.02  males  to  1  female  (grab)  and  0.07  males  to  1  female 
(dredge). 

Results  for  M.  stenolepis  are  primarily  based  on  dredge  data  as  less  than  2% 
were  collected  quantitatively  (Table  I).  M.  stenolepis  was  collected  every  month, 
with  peak  abundances  in  January  and  August.  Ovigerous  forms  were  only  collected 
in  January,  whereas  larvigerous  forms  were  collected  in  January',  March,  and  April. 
The  sex  ratio  of  adults  was  0. 1  males  to  1  female. 

Relationship  to  bathymetry 

Most  N.  americana  in  Cape  Cod  Bay  were  collected  in  shallow  to  intermediate 
depths  (Table  II).  There  was  a  rapid  decline  in  number  caught  at  depths  greater  than 
40  m.  The  highest  density  occurred  at  30-39  m  and  the  highest  relative  abundance 


MYSIDS  OF  CAPE  COD  BAY 


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482 


D.   MAURER  AND   R.   L.   WIGLEY 


occurred  at  20-29  m.  Peak  density  of  immature  stages  occurred  at  10-19  m  and 
peak  relative  abundance  of  immature,  ovigerous,  and  larvigerous  stages  was  at  0.19 
m  (Table  II).  The  frequency  and  relative  abundance  of  N.  americana  (dredge)  de- 
creased significantly  (a  =  0.01)  with  depth  (R  =  -0.56,  R  =  -0.35)  as  did  the  fre- 
quency in  grab  samples  (R  =  —0.29,  a  ;=  0.05). 

Maximum  numbers  of  E.  erythropthalma  were  collected  in  intermediate  depths 
in  Cape  Cod  Bay  (Table  II).  There  was  a  marked  increase  in  numbers  at  depths 

TABLE  II 
Relationship  to  bathymetry  (m)  of  common  mysids  by  sex  and  stage  in  Cape  Cod  Bay. 


Species 

Grab  (No./nr) 

Dredge  (No.  /haul) 

Grand 
total 

Bathymetric 

Range  (m)        M 

F 

Imm 

Lar 

Total 

M 

F 

Imm 

Ovig 

Lar 

Total 

N.  americana 

0-9                30 

41 

53 

1 

125 

121 

166 

58 

1 

11 

357 

482 

10-19              17 

60 

127 

1 

205 

127 

265 

136 

11 

34 

573 

778 

20-29              37 

103 

25 

0 

165 

445 

507 

96 

0 

4 

1052 

1217 

30-39            142 

176 

27 

0 

345 

186 

66 

1  1 

0 

0 

263 

608 

40-49               2 

5 

1 

0 

8 

11 

15 

3 

0 

0 

29 

37 

50-59               0 

0 

0 

0 

0 

1 

0 

0 

0 

0 

1 

1 

Total             228 

385 

233 

2 

848 

891 

1019 

304 

12 

49 

2275 

3123 

E.  erythropthalma 

0-9                 0 

2 

0 

0 

2 

1 

0 

0 

0 

0 

1 

3 

10-19                2 

6 

1 

0 

9 

10 

43 

2 

4 

2 

61 

70 

20-29              30 

59 

21 

1 

111 

162 

240 

88 

0 

0 

490 

601 

30-39                7 

48 

14 

1 

70 

211 

354 

91 

1 

9 

666 

736 

40-49              1  7 

58 

37 

0 

112 

45 

134 

18 

0 

0 

197 

309 

50-59                5 

15 

1 

(1 

21 

10 

35 

10 

1 

1 

57 

78 

Total               6  1 

188 

74 

2 

325 

439 

806 

209 

6 

12 

1472 

1797 

M.  mixla 

0-9                 0 

0 

0 

0 

0 

0 

2 

1 

0 

0 

3 

3 

10-19               0 

13 

14 

0 

27 

0 

47 

6 

0 

8 

61 

88 

20-29               0 

15 

1 

0 

16 

16 

98 

7 

2 

14 

137 

153 

30-39               0 

25 

2 

0 

27 

12 

342 

142 

0 

4 

500 

527 

40-49                2 

27 

1 

0 

30 

16 

105 

59 

3 

31 

214 

244 

50-59               0 

4 

0 

0 

4 

11 

32 

0 

3 

0 

46 

50 

Total 

84 

18 

0 

104 

55 

626 

215 

8 

57 

961 

1065 

M.  stenolepis 

0-9                 0 

0 

0 

0 

0 

3 

19 

0 

0 

1 

23 

23 

10-19               0 

2 

0 

0 

2 

5 

53 

0 

0 

5 

63 

65 

20-29               0 

0 

0 

0 

9 

31 

0 

4 

5 

49 

49 

30-39               0 

0 

0 

0 

0 

1 

6 

0 

1 

1 

9 

9 

40-49               0 

.1 

0 

0 

2 

2 

0 

2 

1 

7 

7 

50-59               0 

0 

(1 

0 

0 

0 

0 

0 

0 

0 

0 

Total                     0 

2 

0 

0 

2 

20 

1  1  1 

0 

7 

13 

151 

153 

M  =  male;  F  =  female;  Imm  =  immature;  Ovig  =  ovigerous;  Lar  =  larvigerous. 
No  ovigerous  individuals  were  collected  by  the  grab  method. 


MYSIDS  OF  CAPE  COD  BAY  483 

greater  than  20  m.  The  highest  density  occurred  between  20  and  29  m.  Immature 
forms  reflected  the  same  density  distribution  patterns  as  adults.  Ovigerous  and  lar- 
vigerous  stages  (dredge)  were  collected  from  10  to  59  rn  with  a  peak  for  the  latter 
in  the  30-39  m  range.  The  frequency  (grab)  of  this  species  increased  significantly 
(a  ==  0.01)  with  depth  (R  =  0.48). 

Maximum  numbers  ofM.  mixta  were  found  at  middle  depths  (Table  II).  Density 
of  immature  stages  was  highest  at  30-39  m.  Ovigerous  and  larvigerous  stages  also 
tended  to  occupy  middle  depths  (Table  II).  The  frequency  of  M.  mixta  in  grab  (R 
=  0.28)  and  dredge  (R  =  0.27)  samples  increased  significantly  (a.  =  0.05)  with  depth. 

The  depth  range  of  Mysis  stenolepis  resembled  that  of  TV.  americana  more  than 
that  of  the  other  two  common  mysids  (Table  II).  The  highest  relative  abundance 
occurred  at  10-19  m  and  declined  rapidly  at  depths  greater  than  30  m.  Larvigerous 
stages  occurred  from  0  to  49  m  and  ovigerous  stages  occurred  from  20  to  49  m. 
Frequency  and  relative  abundance  decreased  (R  =  -0.33,  R  =  -0.28)  significantly 
(a  ==  0.05)  with  depth. 

Relationship  to  bottom  water  temperature 

Most  N.  americana  were  caught  in  bottom  waters  at  temperatures  between  -1.5 
and  8.1°C  (Table  III).  Numbers  declined  above  8.1°C.  Density  of  immature  stages 
was  highest  between  6.0  and  8.1°C  and  relative  abundance  of  immature  stages  was 
highest  at  8.2  to  12.0°C.  Ovigerous  stages  were  collected  at  8.2  to  12.0°C,  and 
larvigerous  stages  (dredge)  were  sampled  at  temperatures  of  3.3  to  23.5°C.  The 
density  and  relative  abundance  of  TV.  americana  decreased  (R  =  -0.47,  R  =  —0.46) 
significantly  (a.  =  0.01)  with  increasing  temperature. 

Most  E.  erythropthalma  occurred  from  - 1.5  to  8. 1  °C  (Table  III)  with  a  marked 
decline  above  8. 1  °C.  Immature,  ovigerous,  and  larvigerous  stages  were  found  at  the 
same  temperature  range  as  adults.  Density  (R  -0.65),  relative  abundance 
(R  =  -0.54),  and  frequency  (grab  R  :  -0.52,  dredge  R  :  -0.76)  of  E.  erythro- 
pthalma decreased  significantly  («  :=  0.01)  with  increasing  temperature. 

Maximum  densities  of  M.  mixta  occurred  at  temperatures  of  3.3  to  8.1°C, 
whereas  maximum  relative  abundance  occurred  from  -1.5  to  8.1°C  (Table  III). 
Immature  stages  from  both  types  of  collecting  gear  were  most  abundant  from  3.3 
to  8. 1  °C.  In  contrast,  ovigerous  and  larvigerous  stages  (dredge)  were  relatively  more 
abundant  between  -1.5  and  5.9°C.  The  frequency  of  M.  mixta  in  grab  (R  =  -0.38) 
and  dredge  samples  (R  =  -0.67)  decreased  significantly  (a  =  0.05,  a  =  0.01)  with 
increasing  temperature. 

Relative  abundance  of  M.  stenolepis  was  generally  high  throughout  a  range  of 
-1.5  to  23.5°C  (Table  III).  This  was  the  most  eurythermal  species  of  the  common 
Cape  Cod  Bay  mysids.  Immature  stages  were  more  abundant  in  warmer  tempera- 
tures (6.0-23.5°C),  whereas  ovigerous  and  larvigerous  stages  were  more  abundant 
below  6.0°C.  The  frequency  and  relative  abundance  of  M.  stenolepis  decreased  with 
increasing  temperature,  but  the  relationships  were  not  statistically  significant. 

Relationship  to  sediment  type 

Maximum  density  of  TV.  americana  occurred  in  clayey-silt  with  relatively  high 
numbers  in  sand  and  silt  (Table  IV).  This  species  was  also  collected  infrequently 
in  gravel.  Maximum  relative  abundance  occurred  in  sand,  followed  in  decreasing 
order  by  clayey-silt  and  silt,  and  gravel.  Ovigerous  and  larvigerous  stages  were  only 
collected  in  sand.  The  frequency  (dredge)  of  TV.  americana  decreased  with  increasing 


484 


D.    MAURER   AND   R.    L.   WIGLEY 


TABLE  III 
Bottom  water  temperature  distribution  oj  common  mysids  by  se.\  and  stage  in  Cape  Cod  Bay. 


Species 

Temperature 

Range  (°C) 


Grab  (No./m2) 


Dredge  (No./haul) 


M 


Imm      Lar     Total       M 


I  mm      Ovig      Lar     Total 


Grand 
total 


N.  americana 

-1.5-3.2 
3.3-5.9 
6.0-8.1 
8.2-12.0 

12.1-23.5 

Total 


130 
40 

35 
2 

21 


137 
122 

71 
25 
30 


29 

8 

131 
19 
46 


0 
0 

i 
0 

1 


296 

170 

238 

46 

98 


234 

439 

108 

56 

54 


281 

198 

381 

66 

93 


58 
70 
43 
104 
29 


0 
0 
0 
12 
0 


0 
4 

26 
8 

1  1 


573 
711 
558 
246 
187 


228  385   233 


848   891   1019   304 


12    49   2275 


869 
881 
796 
292 
285 

3123 


E  erythroplhalma 

-1.5-3.2  29  51  30  0 

3.3-5.9  16  69  20  1 

6.0-8.1  4  50  21  1 

8.2-12.0  12  16  20 

12.1-23.5  0210 

Total  61  188  74         2 


110 

106 

76 

30 

3 


264 

123 

49 

3 

0 


451 
184 
166 

5 
0 


147 

45 

16 

1 

0 


0 

2 

4 
0 
0 


2 
1 
9 
0 
0 


864 

355 

244 

9 

0 


325       439       806      209 


12        1472 


974 

461 

320 

39 

3 

1797 


M.  mixta 

-1.5-3.2 
3.3-5.9 
6.0-8.1 
8.2-12.0 

12.1-23.5 

Total 


0 
2 
0 
0 
0 


4 

47 

33 

0 

0 

84 


0 

4 

14 
0 

(i 

18 


0 
0 
0 
0 
0 


4 

53 

47 

0 

0 

104 


21 

14 

15 

5 

0 


143 

177 

300 

6 

0 


0 
98 

116 

1 
0 


5 
2 
1 

0 
0 


55        626       215 


48 
9 
0 
0 
0 

57 


217 

300 

432 

12 

0 

961 


221 

353 

479 

12 

0 

1065 


M.  stenolepis 

-1.5-3.2 
3.3-5.9 
6.0-8.1 
8.2-12.0 
12.1-23.5 

Total 


0 
0 

0 
0 
0 

0 


1 

0 

1 

0 
0 


0 
0 
0 
0 
0 


(I 
(I 

0 
0 

I) 


0         0 


1 

0 

1 

0 
0 


14 

1 

4 

1 

0 
20 


14 
15 
34 
20 
28 

111 


0 
0 
0 
0 
0 

0 


7 
0 
0 
0 
0 

7 


4 
9 
0 
0 
0 

13 


39 

25 
38 
21 
28 

151 


40 

25 
39 
21 
28 

153 


M  -  male;  F  =  female;  Imm  =  immature;  Ovig  =  ovigerous;  Lar  =  larvigerous. 
No  ovigerous  individuals  were  collected  by  the  grab  method. 


median  sediment  size  (0)  (R  = -0.64,  a  =  0.01),  increased  with  percent  sand 
(R  =  0.53,  a  =  0.01),  and  decreased  with  percent  clayey-silt  (R  =  -0.51,  a  =  0.01). 
Relative  abundance  also  declined  with  percent  silt  (R  =  —0.37)  and  the  frequency 
(grab)  decreased  with  percent  clayey-silt  (R  =  -0.57,  a  --=  0.01). 

Maximum  density  of  E.  erythropthalma-was  in  clayey-silt  and  silt,  with  immature 
stages  most  abundant  in  clayey-silt  (Table  IV).  Maximum  relative  abundance  oc- 
curred in  silt,  clayey-silt,  ar  d  sand.  Ovigerous  and  larvigerous  stages  were  collected 
throughout  a  range  of  sand  to  clayey-silt.  The  frequency  of  E.  erythroplhalma  in 
grab  (R  =  0.78)  and  dredge  (R  ==  0.72)  samples,  density  (R  =  0.59),  and  relative 
abundance  (R  =  0.66)  increased  significantly  (a  =  0.01)  with  decreasing  0.  The  fre- 


MYSIDS  OF  CAPE  COD  BAY 


485 


TABLE  IV 
Sediment  distribution  of  common  mysids  by  sex  and  stage  in  Cape  Cod  Bay. 


Species 
Sediment 
Type 

Grab  (No./m2) 

Dredge  (No.  /haul) 

Grand 
total 

M         F 

Imm 

Lar 

Total 

M 

F 

Imm 

Ovig 

Lar 

Total 

N.  americana 

gravel 

0          7 

1 

0 

8 

12 

11 

9 

0 

0 

32 

40 

sand 

46        99 

181 

2 

328 

527 

426 

215 

12 

49 

1229 

1557 

silt 

16        63 

22 

0 

101 

208 

516 

66 

0 

0 

790 

891 

clayey-silt 

166      216 

29 

0 

411 

144 

66 

14 

0 

0 

224 

635 

Total 

228      385 

233 

2 

848 

891 

1019 

304 

12 

49 

2275 

3123 

E.  erylhropthalma 

gravel 

0         0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

sand 

2          8 

1 

0 

11 

82 

106 

21 

4 

2 

215 

226 

silt 

20       68 

23 

0 

111 

240 

437 

133 

1 

5 

816 

927 

clayey-silt 

39      112 

50 

2 

203 

117 

263 

55 

1 

5 

441 

644 

Total 

61       188 

74 

2 

325 

439 

806 

209 

6 

12 

1472 

1797 

M.  mixta 


gravel 

0 

5 

2 

0 

7 

0 

0 

0 

0 

0 

0 

7 

sand 

0 

18 

12 

0 

30 

0 

74 

15 

0 

11 

100 

130 

silt 

0 

28 

4 

0 

32 

48 

378 

200 

5 

32 

663 

695 

clayey-silt 

2 

33 

0 

0 

35 

7 

174 

0 

3 

14 

198 

233 

Total 

2 

84 

18 

0 

104 

55 

626 

215 

8 

57 

961 

1065 

M.  slenolepis 

gravel 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

sand 

0 

i 

0 

0 

1 

9 

87 

0 

0 

10 

106 

107 

silt 

0 

1 

0 

0 

1 

8 

11 

0 

5 

3 

27 

28 

clayey-silt 

0 

0 

0 

0 

0 

3 

13 

0 

2 

0 

18 

18 

Total 

0 

2 

0 

0 

2 

20 

111 

0 

7 

13 

151 

153 

M  =  male;  F  =  female;  Imm  =  immature;  Ovig  =  ovigerous;  Lar  =  larvigerous. 
No  ovigerous  individuals  were  collected  by  the  grab  method. 


quency  and  density  increased  significantly  (a  =  0.01)  with  percent  silt  (R  =  0.70, 
R  =  0.52)  and  percent  clayey-silt  (R  =  0.67,  R  =  0.58),  while  relative  abundance 
increased  significantly  (a  =  0.05)  with  percent  silt  (R  =  0.34).  In  contrast,  the  fre- 
quency of  E.  erythropthalma  in  grab  (R  =  -0.50)  and  dredge  (R  =  -0.53)  samples, 
density  (R  =  —0.50),  and  relative  abundance  (R  =  —0.36)  decreased  significantly 
(a  =  0.01,  a  =  0.01,  a  =  0.01,  a  =  0.05)  with  percent  sand. 

Mysis  mixta  occurred  at  greatest  density  in  sand  through  clayey-silt  (Table  IV), 
with  immature  stages  primarily  in  sand.  Maximum  relative  abundance  occurred  in 
silt  followed  by  clayey-silt  and  sand.  Larvigerous  and  ovigerous  stages  were  found 
throughout  a  sand  to  clayey-silt  range.  The  frequency  of  M.  mixta  (dredge)  increased 
significantly  with  increasing  0  (R  =  0.68,  a  =  0.01),  percent  silt  (R  =  0.34,  a 
=  0.05),  and  percent  clayey-silt  (R  =  0.44,  a  =  0.05)  and  decreased  with  percent 
sand  (R  =  -0.51,  a  =  0.01).  The  frequency  of  this  species  (grab)  decreased  signif- 


486  D.   MAURER  AND  R.    L.   WIGLEY 

icantly  (a  :=  0.05)  with  percent  sand  (R  =  -0.28)  and  increased  with  percent  silt 
(R  ==  0.32)  and  percent  clayey-silt  (R  =  0.45.  a  =  0.01).  The  density  decreased  sig- 
nificantly (a  ==  0.01)  with  percent  sand  (R  :  -0.46). 

Most  M.  stenolepis  were  caught  in  sand  (Table  IV).  Larvigerous  stages  were 
relatively  more  abundant  in  sand,  but  ovigerous  stages  were  collected  in  silt  and 
clayey-silt.  The  relative  abundance  of  M.  stenolepis  significantly  increased  (a.  =  0.05) 
with  percent  sand  (R  ;=  0.33)  and  decreased  with  percent  silt  (R  =  -0.42). 

DISCUSSION 

Collecting  gear 

The  grab  sample  data  presented  here  provide  some  of  the  first  quantitative 
estimates  of  densities  of  life  history  stages  of  mysids  in  relation  to  seasonal  and 
environmental  factors  for  the  northeast  United  States.  However,  there  is  some  col- 
lecting bias  between  the  grab  and  dredges.  Dredge  hauls  frequently  collected  more 
life  history  stages,  particularly  ovigerous  and  larvigerous  forms,  than  grab  samples 
(Table  I).  Moreover,  grab  samples  underestimated  the  frequency  and  numbers  of 
M.  stenolepis  (Table  I).  Accordingly  one's  perception  of  mysid  distribution  patterns 
can  be  significantly  affected  by  type  of  collecting  gear  used  (Mauchline.  1980). 

Geographic  distribution 

Erythrops  erythropthalma  and  M.  mixta  are  considered  amphi-Atlantic  species. 
In  contrast,  M.  stenolepis  and  N.  americana  are  considered  warm  temperate  to 
tropical  water  species  ( Wigley  and  Burns,  1971).  Mauchline  ( 1 980)  stated  that  species 
living  south  of  60°N,  including  E.  erythropthalma  and  M.  mixta,  may  intrude  into 
the  Arctic  Ocean  regularly  or  sporadically.  According  to  him  M.  stenolepis  and  N. 
americana  belonged  to  a  fauna  confined  to  the  western  Atlantic  between  60°N  and 
40°N.  Mysis  mixta  was  also  considered  amphi-Atlantic  by  Mauchline  (1980)  but 
also  characteristic  of  coastal  areas.  Occurrence  in  New  England  waters  is  well  doc- 
umented for  species  collected  in  this  study  (Fish,  1925;  Whiteley,  1948;  Wig- 
ley,  1964). 

Seasonal  distribution 

Wigley  and  Burns  (1971)  found  ovigerous  and  larval  stages  of  N.  americana 
from  March  to  October  with  the  largest  numbers  in  March  through  June  and  August 
through  October  along  the  northeastern  U.  S.  continental  shelf  and  slope.  Immature 
stages  were  particularly  numerous  in  August  and  December.  The  situation  in  Cape 
Cod  Bay  differed  in  that  large  pulses  of  adults  occurred  in  February,  April,  and 
December,  ovigerous  stages  occurred  only  in  October,  and  larval  stages  occurred 
from  April  to  October  with  a  May  peak  (Table  I).  Hopkins  (1965)  reported  three 
major  spawning  peaks  of  TV.  americana  (April-May,  June,  August)  in  Delaware  Bay. 
He  encountered  a  few  ovigerous  stages  as  late  as  January  and  February.  Williams 
(1972)  reported  the  greatest  abundance  of  TV.  americana  from  November  to  May 
or  June  in  North  Carolina  estuaries.  He  showed  that  ovigerous  or  larvigerous  stages 
occurred  in  every  month  but  November. 

Mauchline  (1980)  proposed  several  major  types  of  mysid  reproduction  and 
succession  of  generations.  His  classification  included  species  with  0.5,  <1,  1,  2,  3, 
and  >3  generations  per  year.  Neomysis  americana  may  not  fit  easily  into  Mauch- 
line's  (1980)  classification  scheme.  There  is  evidence  to  indicate  that  N.  americana 


MYSIDS  OF  CAPE  COD  BAY  487 

produces  two  generations  a  year  on  Georges  Bank  (Wigley  and  Burns,  1971)  and 
in  Cape  Cod  Bay  (Table  I),  three  in  Delaware  shallow  waters  (Hopkins,  1965),  and 
perhaps  three  or  more  generations  in  North  Carolina  estuaries  (Williams,  1972).  If 
this  pattern  is  accurate,  it  suggests  a  latitudinal  shift  of  reproduction  for  N. 
americana. 

According  to  Wigley  and  Burns  (1971),  ovigerous  stages  of  E.  erythropthalma 
occurred  only  in  August  and  larvigerous  stages  in  August  and  September  along  the 
Atlantic  coast.  This  contrasts  with  our  findings  in  Cape  Cod  Bay  of  ovigerous  stages 
in  May  and  July  and  larvigerous  stages  in  January,  May-September,  and  December 
(Table  I).  These  findings  tend  to  confirm  the  tentative  conclusion  of  a  lengthy 
spawning  period  proposed  by  Wigley  and  Burns  (1971).  Erythrops  erythropthalma 
probably  produces  two  generations  per  year  and  falls  within  Mauchline's  (1980) 
classification. 

Wigley  and  Burns  (1971)  concluded  that  M.  mixta  had  two  definite  age  groups 
in  both  spring  and  fall.  Immature  stages  were  common  in  May  and  October.  No 
ovigerous  specimens  were  present  in  their  collections.  The  only  indication  of  spawn- 
ing season  was  the  presence  of  small  (5.3-6.3  mm)  individuals  in  May,  suggesting 
a  late  winter  or  early  spring  spawning.  Within  Cape  Cod  Bay,  adult,  peaks  occurred 
in  later  winter  and  summer  with  a  July  peak  for  immature  stages  (Table  I).  Tattersall 
( 195 1 )  recorded  many  occurrences  of  adults  in  August  and  September  but  made  no 
references  to  larvigerous  and  ovigerous  stages.  Records  of  ovigerous  stages  in  January 
and  larvigerous  stages  in  January  through  April  (March  peak)  (Table  I)  confirm  the 
late  winter/early  spring  spawning  period  proposed  by  Wigley  and  Burns  ( 197 1 ).  The 
grossly  unbalanced  sex  ratio  reported  for  M.  mixta  from  broad  ranging  samples  on 
the  continental  shelf  was  also  recorded  in  the  more  restricted  confines  of  Cape  Cod 
Bay.  Different  habitat  preferences  for  males  and  females,  different  environmental 
conditions  for  reproduction  and  larval  development,  or  short-lived  life  cycle  for 
males  may  explain  this  pattern. 

Although  data  for  M.  stenolepis  are  sparse,  there  is  a  suggestion  of  peaks  for 
adults  in  winter  (January)  and  summer  (August)  and  for  larvigerous  stages  in  late 
winter/early  spring  (Table  I).  Ovigerous  stages  were  collected  only  in  January.  This 
view  agrees  with  earlier  versions  provided  by  Smith  (1879)  and  Tattersall  (1951). 
According  to  Mauchline's  scheme,  M.  stenolepis  and  probably  M.  mixta  would 
belong  to  species  producing  one  generation  per  year. 

Relation  to  bathymetry 

Mauchline  (1980)  proposed  a  bathymetric  classification  of  mysids  that  included 
recognition  of  the  ecological  significance  of  salinity  (freshwater  and  brackish).  Ex- 
clusive of  brackish  and  freshwater  species,  he  recognized  a  spectrum  ranging  from 
littoral,  to  shallow  shelf,  to  eurybenthic  shelf,  to  bathypelagic. 

Wigley  and  Burns  (1971)  established  five  depth  categories  from  which  mysids 
were  most  frequently  caught.  Neomysis  americana,  E.  erythropthalma,  and  M.  mixta 
were  listed  as  eurybathic  shelf  species  (range  1-421  m),  and  M.  stenolepis  was  cited 
as  a  shore  species  (intertidal).  Within  Cape  Cod  Bay  there  was  evidence  of  spatial 
partitioning  in  terms  of  bathymetric  stratum.  Neomysis  americana  and  M.  stenolepis 
were  characteristic  of  shallow  and  intermediate  depths,  while  E.  erythropthalma  and 
M.  mixta  were  characteristic  of  intermediate  to  greater  depths  (Table  II).  The  as- 
sociations of  N.  americana  and  M.  stenolepis  with  shallow  water  and  of  E.  ery- 
thropthalma with  deeper  water  were  reported  previously  (Segerstrale,  1945;  Tatter- 
sall, 1951,  1954;Bousfield,  1956;  Wigley,  1 964;  Wigley  and  Burns,  1971).  In  contrast, 


488  D.   MAURER  AND  R.   L.   WIGLEY 

Hulburt  (1957)  found  more  N.  americana  at  greater  depths  in  Delaware  Bay.  Their 
low  abundance  in  shallow  water  may  have  been  due  in  part  to  the  presence  of 
caridean  shrimp  (Crangon  septemspinosa  (Say),  Palaemontes  vulgaris  (Say),  P.  pugio 
(Holthuis))  which  are  very  abundant  in  shallow  waters  of  Delaware  Bay  (Price, 
1962). 

The  segregation  of  Cape  Cod  Bay  species  pairs  by  depth  is  indicative  of  zonation, 
which  reduces  competition  for  space.  Zonation  of  nearshore  mysids  (0-17  m)  was 
described  from  a  sand  bottom  on  the  open  coast  of  California  (Clutter,  1967).  He 
concluded  that  zonation  probably  developed  in  response  to  the  availability  of  food 
imposed  by  nearshore  circulation.  This  relationship  cannot  be  ignored  in  Cape  Cod 
Bay;  a  case  for  multivariate  environmental  interaction  is  discussed  later.  In  terms 
of  Mauchline's  (1980)  bathymetric  classification  N.  americana  and  M.  stenolepis  in 
Cape  Cod  Bay  would  fall  within  the  littoral  to  shallow  shelf  habitat  and  E.  ery- 
thropthalma  and  M.  mixta  would  fit  the  shallow  shelf  to  eurybenthic  shelf  habitat. 

Relationship  to  bottom  water  temperature 

Neomysis  americana  is  considered  to  be  eurythermic,  found  at  bottom  water 
temperatures  from  0  to  25 °C  (Wigley  and  Burns,  1971).  Within  Cape  Cod  Bay  N. 
americana  occurred  throughout  a  similar  temperature  range,  but  their  maximum 
distribution  was  between  —1.5  and  8. 1  °C  (Table  III).  Specimens  from  Delaware  Bay 
were  most  abundant  at  lower  temperatures  (Hulburt,  1957).  Erythrops  erythro- 
pthalma  showed  a  bottom  water  temperature  distribution  similar  to  that  of  N. 
americana,  but  its  abundance  peaked  in  even  lower  temperatures  (—1.5  to  3.2°C) 
(Table  III).  Mysis  stenolepis  occurred  throughout  the  local  temperature  range,  but 
M.  mixta  was  only  collected  below  12.0°C  (Table  III).  The  peak  of  the  latter  species 
was  6.0-8.1°C.  The  former  species,  together  with  N.  americana,  occurred  in  ap- 
preciable numbers  above  12.0°C.  The  local  temperature  occurrence  of  the  two  spe- 
cies of  Mysis  was  consistent  with  their  shallow  and  deeper  water  habits. 

Bottom  water  temperature  changes  seasonally.  However,  there  was  evidence  for 
interaction  between  temperatures  and  depth  on  abundance  and  frequency  of  mysids. 
Maximum  abundance  of  N.  americana,  E.  erythropthalma,  and  M.  stenolepis  oc- 
curred in  a  temperature  range  of  —  1 .5  to  8. 1  °C,  which  coincided  with  high  seasonal 
numbers  recorded  for  January  through  March.  A  similar  relationship  can  be  seen 
for  M.  mixta  with  maximum  abundance  in  6.0-8.1°C  which  coincided  with  high 
seasonal  numbers  recorded  for  May  and  July.  Seasonal  effects  are  evident  in  these 
relationships. 

However,  the  relationship  between  bottom  water  temperature  and  the  deeper 
water  mysid  pair  (E.  erythropthalma  and  M.  mixta)  was  further  complicated  by 
depth-temperature  interaction.  A  marked  summer  thermocline  was  reported  in  Cape 
Cod  Bay  from  mid-April  until  Mid-October  (Young  et  al.,  1971).  The  annual  tem- 
perature at  20  to  26  m  ranged  from  -1.5  to  10°C.  The  lowest  extent  of  the  ther- 
mocline defined  by  the  5°C  isotherm  intersected  the  sea  floor  at  approximately  26 
m.  Even  though  mysid  distribution  is  influenced  by  seasonal  effects  of  bottom  water 
temperature,  the  latter  is  influenced  by  bathymetry.  It  might  be  expected  that  the 
shallow  water  pair  of  mysids  are  more  responsive  to  seasonal  water  temperatures, 
whereas  the  deeper  water  pair  are  more  affected  by  depth-bottom  temperature  re- 
lationships. Emberton  (1981)  showed  that  selected  taxa  of  subtidal  meiofauna  in 
Cape  Cod  Bay  were  significantly  influenced  by  season-depth  interactions.  Season 
was  more  important  in  shallow  water,  whereas  there  was  a  time  lag  at  greater  depths 
in  terms  of  meiofauna  density. 


MYSIDS  OF  CAPE  COD  BAY  489 

Relationship  to  sediment  type 

Mauchline  (1980)  cited  many  cases  of  sediment  preference  for  hyperbenthic 
mysids.  Wigley  and  Burns  (1971)  summarized  the  sediment  distribution  of  mysids 
as  follows:  N.  americana  and  E.  erythropthalma  on  sand,  M.  stenolepis  on  sand  and 
Zostera,  and  M.  mixta  on  a  variety  of  sediments.  However,  the  present  study  showed 
that  silt  and  clayey-silt  played  an  important  role  in  the  distribution  of  Cape  Cod 
Bay  mysids  (Table  IV).  Williams  (1972)  cited  evidence  that  N.  americana  in  North 
Carolina  estuaries  commonly  occurred  over  sediments  of  clay  and  silt-sized  particles. 
Young  and  Rhoads  (1971)  collected  quantitative  samples  in  Cape  Cod  Bay  and 
reported  N.  americana  from  sand  and  clayey-silt. 

In  this  study,  N.  americana  had  a  wide  sediment  range  occurring  in  gravel 
through  clayey-silt  (Table  IV).  Even  though  maximum  abundance  (dredge)  was 
reported  in  sand,  relatively  high  numbers  were  also  recorded  in  silt  and  clayey-silt. 
Moreover,  maximum  density  was  recorded  from  clayey-silt.  This  broad  sediment 
range,  together  with  its  eurythermic  characterization  and  broad  salinity  range  (Hul- 
burt,  1957),  is  consistent  with  its  occupancy  of  coastal  areas  and  estuaries  which 
normally  display  rapidly  changing  environmental  conditions.  The  detrital  load  of 
estuaries  and  the  feeding  habit  of  N.  americana  are  also  involved  in  this  association. 

In  view  of  the  bias  toward  sand  samples  in  the  collection,  maximum  abundance 
and  occurrence  of  E.  erythropthalma  in  silt  and  clayey-silt  indicates  the  importance 
of  this  sediment  type.  This  relationship  is  supported  by  other  findings  (Young  and 
Rhoads,  1971).  Thus,  the  earlier  view  of  the  sediment  distribution  of  E.  erythro- 
pthalma as  characteristically  occurring  in  sand  (Wigley  and  Burns,  1971)  should  be 
amended  to  include  bottoms  with  significant  amount  of  silt  and  clayey  silt. 

Mysis  mixta  had  a  broad  sediment  range  comparable  to  N.  americana  but  oc- 
curred most  abundantly  in  Cape  Cod  Bay  in  silt  and  clayey-silt  (Table  IV).  This 
distribution  is  generally  consistent  with  an  earlier  view  (Wigley  and  Burns,  1971). 
Mysis  stenolepis  peaked  in  sand  in  Cape  Cod  Bay,  but  this  species  may  live  in 
sediment  containing  as  high  as  18%  silt  or  12%  clayey-silt.  It  appears  that  fine  grain 
sediment  can  be  considerably  more  important  in  the  ecology  of  these  four  mysids 
than  previously  recognized.  The  relationships  among  fine  sediment,  paniculate  or- 
ganics,  microbiota,  and  mysid  feeding  habits  deserve  attention  (Mauchline,  1980) 
because  M.  stenolepis  may  be  able  to  digest  cellulose  (Wainwright  and  Mann,  1982). 

There  was  evidence  of  significant  relationships  between  mysid  frequency,  den- 
sity, and  relative  abundance  and  sediment  type.  Sediment  decreases  in  modal  size 
and  increases  in  total  clay  and  carbon  contents  with  depth  in  Cape  Cod  Bay,  and 
seston  flux  is  10  times  greater  at  the  deeper  muddy  stations  than  at  the  shallow 
sandy  stations  (Young  et  al.,  1971).  Young  et  al.  reported  difficulty  in  determining 
which  environmental  factor  was  most  important  in  separating  zones  of  polychaetes 
in  Cape  Cod  Bay  because  the  isopleths  of  10°C,  15-20  m,  and  20%  mud  closely 
coincided.  Their  findings  are  consistent  with  the  view  of  interactions  among  bottom 
water  temperatures,  depth,  and  sediment  distribution.  Accordingly,  distributions  of 
mysid  species  are  probably  influenced  by  these  interactions.  Since  the  same  inter- 
actions are  not  as  well  defined  along  the  northeastern  part  of  the  bay,  seasonal- 
temperature  factors  may  be  more  important  here. 

In  summary,  zonation  of  these  mysids  was  related  to  depth-temperature-sedi- 
ment interactions  within  a  seasonal  framework.  These  multi factorial  environmental 
effects  were  expressed  by  a  shallow  water,  silt-sand  to  sand  pair  of  mysids  (N. 
americana  and  M.  stenolepis}  and  a  deeper  water,  silt  to  clayey-silt  pair  (E.  ery- 
thropthalma and  M.  mixta).  Evidence  for  partitioning  related  to  biotic  factors  was 


490  D.   MAURER   AND  R.   L.   WIGLEY 

not  included  in  this  study,  but  evidence  of  considerable  predation  on  mysids  by 
finfish  and  competition  through  co-occurrences  of  mysids  has  been  presented  else- 
where (Wigley  and  Burns,  1971;  Mauchline,  1980).  In  general,  these  mysids  fall 
within  Mauchline's  (1980)  bathymetric  and  reproductive  classifications,  with  dif- 
ferences from  these  distributions  associated  with  regional  and  seasonal  conditions. 

LITERATURE  CITED 

BIGELOW,  H.  B.,  AND  M.  SEARS.  1939.  Studies  of  the  waters  of  the  continental  shelf.  Cape  Cod  to 

Chesapeake  Bay.  III.  A  volumetric  study  of  the  zooplankton.  Mem.  Mus.  Comp.  Zoo/.  Harvard 

Coll.  LIV(4):  189-378. 

BOUSFIELD,  E.  L.  1956.  Studies  on  the  shore  Crustacea  collected  in  eastern  Nova  Scotia  and  Newfound- 
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BOUSFIELD,  E.  L.  1961.  Studies  on  littoral  marine  arthropods  from  the  Bay  of  Fundy  region.  Can.  Dept. 

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Reference:  Biol.  Hull.  163:  492-503.  (December  1982) 


MEMBRANE-STABILIZING  AND  CALCIUM-BLOCKING  AGENTS 
AFFECT  ARBACIA   SPERM   MOTILITY 

LEONARD  NELSON 

Department  of  Physiology.  Medical  College  of  Ohio,  Toledo,  OH  43699,  and 
Marine  Biological  Laboratory,  Woods  Hole.  MA  02543 

ABSTRACT 

The  speed  and  duration  of  progressive  motility  of  Arbacia  sperm  cells  depend 
on  the  calcium  content  of  the  suspension  medium.  Suspended  in  filtered  sea  water 
(FSW)  the  spermatozoa  undergo  a  progressive  decline  in  motility  (after  an  initial 
burst  of  activity)  and  cease  forward  movement  within  30-40  min.  When  sperm  are 
diluted  in  chemically  defined  artificial  sea  water  (MBL-ASW),  motility  rose  to  about 
160%  of  the  control  rate  in  30  min  and  then  gradually  returned  to  the  initial  control 
level  where  it  persisted  for  at  least  40  min  more.  Procaine,  propranolol.  ouabain, 
and  quinidine,  tested  singly  or  in  combination,  affected  sperm  motility  in  both  time- 
and  concentration-dependent  fashion. 

Procaine  at  10  and  100  n\l/\  in  MBL-ASW  caused  more  than  a  doubling  in 
motility  over  the  control  rate,  while  in  FSW  both  these  concentrations  were  inhib- 
itory. In  FSW,  quinidine  had  relatively  little  effect,  while  propranolol  was  slightly 
stimulatory  at  10~6M  and  inhibitory  at  0.1  and  l.OX  10  3  M.  In  combination, 
propranolol  and  quinidine  can  cause  a  sharp  rise  in  motility.  Ouabain  increased 
motility  dramatically  in  MBL-ASW  suspensions.  The  effects  of  some  of  the  drugs 
depend  on  the  ability  to  displace  calcium  from  binding  sites  in  sperm  cell  mem- 
branes; ouabain  appears  to  interface  with  Ca  efflux. 

INTRODUCTION 

Receptor  activation  and  membrane  lability  play  critical  roles  in  the  activity  of 
many  cell  types.  For  example,  induction  of  platelet  aggregation  by  specific  agonists 
is  inhibited  by  substances  classified  as  local  anesthetics  and  antiarrhythmic  agents; 
calcium  antagonizes  these  inhibitory  actions  (Anderson  el  al..  1981).  Further,  the 
effects  of  Ca2+  in  the  medium  on  ciliary  beat  reversal  in  paramecium  has  been  amply 
documented  (Murakami  and  Eckert,  1972). 

Similarly  the  movement  of  the  mature  spermatozoa  of  mammals  and  marine 
invertebrates  is  greatly  influenced  by  interactions  between  sperm  cell  components 
and  environmental  factors.  Responsiveness  of  sperm  cell  receptors  to  ligands,  ac- 
tivators, and  inhibitors,  appears  to  vary  with  the  condition  of  the  sperm  cell,  its  state 
of  dilution  (Gray,  1928;  Rothschild,  1953),  maturation  (Babcock  el  al.,  1979),  aging 
(Dunham  el  al.,  1982),  capacitation,  and  even  proximity  to  the  ovum  (Yanagi- 
machi,  1970). 

In  the  presence  of  some  agents,  other  conditions  being  equal,  the  rate  of  sperm 
cell  propulsion  increases  considerably.  This  implies  that,  under  usual  circumstances, 
not  all  of  the  sperm  cells  in  a  given  sample  are  progressing  at  their  maximal  speed; 

Received  28  January  1982;  accepted  7  September  1982. 

Abbreviations:  EDTA,  ethylene  diamine  tetra  acetate;  EGTA,  ethylene  glycol  bisaminoethyltetra 
acetate;  FSW,  filtered  sea  water;  MBL-ASW,  Marine  Biological  Laboratory  formulated  artificial  sea  water. 
This  work  supported  by  NSF  Grant  #PCM8002358. 

492 


CA-BLOCKERS  AND  SPERM   MOTILITY  493 

that  is,  there  seems  to  be  a  margin  of  safety  which  may  permit  the  conservation  of 
energy  stores  or  otherwise  enhance  the  union  of  physiologically  uncompromised 
gametes.  The  difference  between  the  optimum  and  the  maximum  swim  speed  ca- 
pacity suggests  the  presence  in  the  sperm  cell  of  a  regulatory  mechanism  modulated 
by  control  of  calcium  entry  and  transport  through  the  cell  as  proposed  here. 

Procaine,  added  to  sea  water  suspensions  of  Arbacia  sperm,  caused  a  rapid  rise 
in  their  mean  rate  of  forward  motion  followed  by  a  sharp  decline  (Nelson,  1972). 
The  local  anesthetic  apparently  occupied  binding  sites  in  the  plasma  membrane, 
and  having  driven  some  of  the  bound  calcium  into  the  cell  interior,  then  prevented 
its  efflux. 

The  critical  role  of  Ca2+  in  the  modulation  of  sperm  motility  was  further  em- 
phasized in  studies  with  ZnCl2,  MnCl2,  and  EDTA  (Young  and  Nelson,  1974a)  and 
CaCl2,  LaCl3,  and  EGTA  (Young  and  Nelson,  1974b).  Zn2+  and  Ca2+  had  distinctly 
biphasic  effects,  while  Mn2+  and  La3+  as  well  as  EDTA  and  EGTA  were  inhibitory 
or  ineffective  in  the  concentration  ranges  tested. 

Cholinergic  mediation  appears  to  be  involved  in  regulation  of  the  entry  of  cal- 
cium into  the  sperm  cell  through  specific  ion  channels  (Nelson  et  al.,  1980).  That 
is,  calcium  transport  seems  to  depend  on  acetylcholine-induced  conformational 
changes  in  a  receptor  channel  complex  that  extends  through  the  plasma  membrane, 
similar  to  that  proposed  by  Cohen  and  Changeux  (1975)  for  cationic  transport  at 
myoneural  junctions  and  electroplaques. 

Arbacia  sperm  cells  respond  to  nicotine,  maximum  stimulation  occurring  at 
1 0"9  M  and  inhibition  commencing  at  1 0~6  M.  The  highly  selective  nicotinic  receptor 
blocker,  a-bungarotoxin,  completely  inhibits  all  the  cells  in  a  suspension  of  Arbacia 
sperm  at  less  than  10~6  M;  microscopic  examination  showed  that  individual  cells 
ceased  moving  at  a  concentration  of  less  than  1  picomole/1  (Nelson,  1976). 

In  mammalian  sperm  catecholamine  sensitivity  appears  at  the  onset  of  the  cy- 
tostructural  and  permeability  changes  coincident  with  capacitation  (Bavister  et  al., 
1976;  Cornett  and  Meizel,  1978),  but  neither  epinephrine  nor  norepinephrine  was 
observed  to  exert  any  appreciable  change  in  the  swim  speed  of  Arbacia  sperm  that 
had  not  been  exposed  to  capacitating  conditions  (unpublished  observations). 

Local  anesthetics  that  block  nerve  conduction  and  have  pronounced  effects  on 
muscle  contraction  raise  the  threshold  for  osmotic  hemolysis  of  erythrocytes  and 
interfere  with  platelet  aggregation.  The  action  of  procaine  on  Arbacia  spermatozoa 
attests  to  excitability  as  a  physiological  characteristic  of  the  regulatory  processes 
governing  the  movement  of  these  cells. 

This  report  extends  studies  on  the  effects  of  procaine  on  sea  urchin  sperm  (Nel- 
son, 1972)  to  include  an  examination  of  the  action  of  the  /3-adrenergic  blocking 
agent  propranolol  and  the  a-blocker  quinidine  which  when  applied  directly  to  car- 
diac muscle  exerts  an  action  similar  to  that  of  procaine.  Propranolol  is  effective  in 
combatting  cardiac  glycoside  intoxication,  and  so  the  interactive  effects  of  propran- 
olol and  ouabain  on  sperm  motility  were  also  examined. 

MATERIALS  AND  METHODS 

Semen  was  collected  daily  from  mature  Arbacia  punctulata  induced  to  spawn 
by  injection  of  1  ml  of  0.5  M  KC1  through  the  oral  surface  of  the  animal.  The  sea 
urchins  were  inverted  over  30-ml  beakers  filled  with  either  filtered  sea  water  (FSW) 
or  chemically  defined  Marine  Biological  Laboratory  artificial  sea  water  (MBL-ASW). 
(The  dense  semen  streams  settled  rapidly  and  coherently  to  the  bottom  of  the  beaker 
without  dispersing).  This  procedure  permits  the  preparation  of  samples  from  the 


494  LEONARD  NELSON 

same  sea  urchin  for  suspension  in  either  FSW  or  MBL-ASW,  for  use  for  an  entire 
series  of  experimental  runs.  The  supernatant  fluid  was  decanted  and  the  concentrated 
sperm  cells  were  aspirated  and  transferred  by  means  of  disposable  Pasteur  pipettes 
into  test  tubes  kept  in  an  ice  bath  for  the  day's  tests.  As  needed,  sufficient  concen- 
trated sperm  was  diluted  in  25  ml  of  FSW  or  MBL-ASW  to  yield  an  optical  density 
reading  between  0.500  and  0.700  in  a  Turner  Model  350  Spectrophotometer 
(A  ==  480  nm),  equivalent  to  7-10  >  106  sperm/ml  (Nelson,  1972). 

For  each  experiment,  different  concentrations  of  the  test  reagents  were  quan- 
titatively added  by  micropipet  to  6  separate  round  cuvettes  and  the  volume  brought 
to  0.5  ml  with  FSW  or  MBL-ASW.  The  tests  were  initiated  by  addition  of  2.0  ml 
of  the  sperm  suspension  diluted  immediately  prior  to  the  start  of  each  run.  The 
"zero-time"  reading  was  taken  in  the  Spectrophotometer  after  first  mixing  the  cuvette 
contents  by  twice  inverting  the  parafilm-covered  tube.  The  cuvettes  were  then  put 
into  the  six-place  horizontal  rotor  of  an  I. E.G.  clinical  centrifuge  and  spun  for  4 
minutes  at  120  X  g  (940  rpm);  this  has  empirically  been  shown  to  align  the  sper- 
matozoa with  only  minimal  centrifugal  sedimentation  of  non-motile  cells  (ibid.}. 

Orientation  of  the  spermatozoa  subjected  to  low  centrifugal  force  permits  re- 
producible measurement  of  changes  in  optical  density  of  the  suspensions  as  the  cells 
swim  past  the  light  path.  As  the  cells  are  stimulated,  depressed,  or  unaffected  by 
varying  concentrations  of  a  given  combination  of  agents,  optical  density  differences 
between  the  untreated  controls  and  the  treated  suspensions  are  recorded  from  the 
Spectrophotometer.  The  difference  in  O.D.  between  the  zero  time  and  4-min  cen- 
trifugal runs  of  the  various  specimens  (after  correction  for  displacement  of  formalin- 
killed  cells,  if  any,  is  made)  is  determined.  All  the  tubes  in  that  series  are  normalized 
to  the  4-min  control  reference  point,  as  percent  of  control  motility.  Motility  refers 
to  progressive  motion.  (For  full  details,  see  Nelson,  1972.)  All  of  the  test  reagents 
employed — procaine  (free  base);  ouabain  •  8H2O;  DL-propranolol  •  HC1,  and  quin- 
idine  •  SO4 — were  of  the  purest  grade  available  from  Sigma  Chemical  Company. 
Artificial  sea  water  (MBL-ASW)  prepared  in  the  Chemical  Department  of  the  Marine 
Biological  Laboratory,  Woods  Hole,  MA,  contained  (in  mAI  per  liter  of  deionized 
water):  NaCl,  423;  KC1,  9.0;  CaCl2-2H2O,  9.27;  MgCl2,  22.94;  MgSO4,  25.50;  just 
prior  to  use  0.18  mg  NaHCO3/l  was  added.  The  inorganic  salts  were  of  analytical 
reagent  grade,  meeting  ACS  specifications. 

All  experiments  were  conducted  at  room  temperature  which  ranged  from  22.5° 
to  25 °C  during  the  course  of  the  season. 

RESULTS 

This  group  of  test  agents  was  selected  because  Ca2+  has  been  implicated  as  a 
second  messenger  in  cellular  responses  to  their  action.  The  swimming  capacity  of 
Arbacia  sperm  cells  has  long  been  known  to  deteriorate  within  30-60  minutes  after 
dilution  in  sea  water.  This  so-called  "dilution  effect"  starts  with  sharp  increases  in 
oxygen  uptake  and  rate  of  movement  which  presumably  rapidly  deplete  energy 
stores.  Figure  1  illustrates  the  loss  of  motility  of  the  FSW-diluted  sperm  cells,  drop- 
ping to  zero  within  40  minutes.  The  abrupt  rise  in  activity  was  not  evident  in  these 
determinations  since  the  first  motility  rating  was  not  scored  until  five  to  six  minutes 
after  dilution.  In  the  sperm  samples  suspended  in  MBL-ASW  a  protracted  rise  in 
the  motility  rate  occurs  that  peaks  at  about  20  minutes  and  returns  to  the  initial 
level  for  the  duration  of  the  experiment.  When  the  sperm  cells  are  suspended  in  a 
90:10  mixture  of  MBL-ASW:FSW,  the  rate  of  increase  and  the  maximum  rate  are 
both  reduced  and  the  sperm  cell  motility  then  gradually  drops  down  to  about  half 
the  speed  in  the  MBL-ASW  alone. 


CA-BLOCKERS  AND  SPERM   MOTILITY 


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Duration  (mini 

FIGURE  1 .  Dilution  effects:  dependence  of  rate  and  duration  ofArbacia  sperm  swimming  on  calcium 
content  of  the  suspension  medium:  a)  open  circles,  MBL  artifical  sea  water  (MBL-ASW);  b)  half-circles, 
90:10  mixture  of  MBL-ASW:FSW;  c)  closed  circles,  filtered  sea  water  (FSW).  Ordinate,  relative  speed 
of  sperm  cell  progression  (as  percent  of  control)  following  dilution:  abscissa,  time  elapsed  after  dilution. 
Sperm  cells  in  MBL-ASW  attain  higher  speeds  and  endure  for  longer  periods  than  those  in  filtered  sea 
water.  Temp.  22.5°C. 


In  previous  studies,  the  immediate  effects  of  several  concentrations  of  calcium 
and  of  procaine  were  examined.  The  present  results  with  procaine  indicate  that  both 
time  dependence  and  concentration  dependence  of  the  response  are  modulated  by 
the  relative  amounts  of  contaminants  (presumably  traces  of  heavy  metals)  in  the 
medium.  Figures  2a  (FSW)  and  2b  (MBL-ASW)  show  close  replication  of  the  re- 
spective controls  (no  procaine)  between  the  duration  of  sperm  cell  exposure  to 
filtered  sea  water  and  the  artificial  sea  water  demonstrated  in  Fig.  1.  In  the  presence 
of  procaine,  the  sperm  cells  in  FSW  (Fig.  2a)  generally  undergo  a  fairly  precipitous 
decline  in  motility,  paralleling  the  control  curve;  10  2  M  procaine  is  predictably 
inhibitory  from  the  start,  the  lower  concentrations  not  differing  significantly  from 
the  controls.  With  MBL-ASW  as  the  suspending  medium  (Fig.  2b),  even  the  10~2 
M  procaine  shows  an  initial,  pronounced,  increased  acceleratory  effect,  the  swim- 
ming speed  rising  to  about  1 70%  of  the  control  rate  in  1 5  minutes.  The  speed  returns 
to  the  control  levels  by  20  minutes  and  then  approaches  a  plateau  while  the  controls 
continue  their  downward  rate.  Sperm  cells  suspended  in  millimolar  procaine  closely 
parallel  the  controls  for  the  first  twenty  minutes  but  then  decline  much  less  abruptly. 
The  spermatozoa  in  10~5  and  10~4  M  procaine,  however,  peak  at  nearly  double  the 
control  speed  in  ten  minutes  and  decrease  gently  to  a  level  of  forward  motion  3-4 
times  greater  than  that  of  the  untreated  controls. 

Procaine  acts  at  the  sperm  cell  surface;  purportedly  it  affects  cationic  channels 
involved  in  calcium  entry  by  displacing  calcium  from  binding  sites.  Conversely 
ouabain,  a  specific  inhibitor  of  Na+,  Reactivated,  Mg2+-dependent  adenosinetri- 


496 


LEONARD  NELSON 


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80 


Minutes 


FIGURE  2.  Effects  of  procaine  on  motility  of  Arbacia  sperm  suspended  in  a)  FSW  and  b)  MBL- 
ASW.  Ordinate,  percent  of  control  motility  rate;  abscissa,  time  elapsed  following  dilution.  Note  that 
procaine-treated  sperm  suspended  in  MBL-ASW  swim  at  a  higher  rate  of  speed  for  longer  periods  than 
do  similarly  treated  sperm  in  FSW. 

phosphatase,  is  considered  to  impede  Ca2+  efflux  from  cardiac  muscle  cells  (Wood 
et  al.,  1972)  and  may  similarly  affect  sperm  cells  (cf.  review,  Nelson  and  McGrady, 
1981).  The  maximum  effect  of  a  10-min  incubation  in  ouabain  in  filtered  sea  water 
occurs  at  about  10~6  M.  This  is  shown  in  Figure  3  and  confirms  the  previous  report 
(Nelson,  1972).  Increasing  the  incubation  periods  in  MBL-ASW  shifts  the  maximum 
response  to  the  left;  incubation  in  10~9  M  ouabain  produces  a  peak  in  30  minutes. 
This  is  again  a  dramatic  (2.5  fold)  increase  over  that  of  the  initial  rate  of  the  FSW 
controls. 

Ouabain  toxicity  in  the  mammalian  heart  cell  may  be  counteracted  by  the  beta- 
adrenergic-blocking  agent  propranolol  (which  in  itself  exhibits  some  of  the  Ca-per- 
turbing  properties  of  a  local  anesthetic).  Propranolol  and  ouabain  were  therefore 
assayed  singly  and  in  combination;  their  interactive  effects  were  tested  on  the  pro- 
gressive motility  of  sperm  in  filtered  sea  water.  Lower  concentrations  of  propranolol 
have  little  effect  except  for  a  20%  increase  at  10  6  M  (Fig.  4).  However,  at  0.1  mM 
a  40%  decrease  in  motility  occurs,  while  the  inhibition  increases  to  80%  at  1  mM. 
The  peak  effect  of  ouabain  alone  (at  10~6  M)  was  a  65%  increase  in  the  swimming 
rate  over  the  controls.  In  the  optimum  concentration  of  ouabain  ( 10  6  M),  increasing 
amounts  of  propranolol  tend  to  lower  the  motility  response  curve  by  about  5-10%. 
Above  the  optimum  concentration  of  both  drugs  (10~6  M  each),  the  ouabain  did 
not  significantly  influence  the  response  to  propranolol.  However,  with  ouabain  set 
a  concentration  of  10~3  M  throughout,  the  responses  to  varying  amounts  of  pro- 
pranolol are  markedly  altered.  Both  the  prominent  peak  at  10~6  Mand  the  profound 
depression  at  higher  propranolol  concentrations  are  eliminated. 

Cinchona  alkaloids  reportedly  exhibit  digitalis-like  properties.  Therefore  a  fur- 
ther test  of  propranolol  in  drug-interactive  effects  on  the  sperm  cell's  ability  to  swim 
progressively  is  afforded  in  the  experiments  with  quinidine.  As  in  the  preceding 


CA-BLOCKERS  AND  SPERM  MOTILITY 


497 


150- 


-»  x 


p  Ouabain 

FIGURE  3.  Time-  and  dose-dependent  effects  of  ouabain  on  Arbacia  sperm  in  FSW  and  MBL- 
ASW.  X's,  after  10  min  exposure  in  FSW.  Open  symbols,  in  MBL-ASW:  circles,  10  min;  triangles,  20 
min;  squares,  30  min  after  dilution.  Ordinate,  relative  motility  in  percent  of  control  rate;  abscissa,  negative 
log  of  molar  concentration  of  ouabain  in  the  medium. 


experiment  (Fig.  4),  the  peak  in  10  6  M  propranolol  is  succeeded  by  a  sharp  motility 
decline  with  increasing  drug  concentration.  Figure  5a  shows  that  prolonging  the 
incubation  in  propranolol  has  little  added  effect  at  the  lower  concentrations,  but  at 
micromolar  amounts  stimulation  becomes  evident  and  inhibition  is  somewhat  ame- 
liorated at  concentrations  of  10~5  M  and  higher. 

Incubation  in  quinidine  alone  in  FSW  (shown  in  Fig.  5b)  over  a  range  of  con- 
centrations from  10~'°  M  to  10~4  M  evokes  a  somewhat  uneven  but  insignificant 
oscillation  around  the  control  rate  of  movement.  The  delayed  effect  on  motility 
does  not  deviate  strikingly  from  that  of  the  delayed  controls  when  the  "dilution" 
effect  is  taken  into  account,  viz.,  a  60%  to  80%  decrease  in  progressive  movement 
which  is  sustained  over  the  entire  concentration  range  after  an  additional  ten  minutes 
of  incubation.  When  the  two  drugs  are  tested  for  interactive  effects,  the  samples 
incubated  in  10"3  M  quinidine  responded  more  vigorously  than  those  in  10"5  M. 
In  these  experiments,  the  Arbacia  sperm  cells  in  seawater  suspension  were  prein- 
cubated  for  five  minutes,  and,  after  their  motility  was  rated,  to  each  cuvette  was 
added  0.2  ml  FSW  in  the  single  treatment  labeled  "Q"  or  "P"  or  0.2  ml  of  quinidine 
for  the  co-incubation,  double-treatment  series,  labeled  "P  +  Q"  in  the  bar  graph 
diagrams  (Figs.  6a  &  6b).  After  the  additions,  the  cuvettes  were  again  inverted  2 
times  to  assure  uniform  redistribution.  The  sperm  cells  were  then  reoriented  cen- 
trifugally,  and  readings  were  taken  at  the  indicated  intervals.  Sperm  cells  exposed 


498 


LEONARD  NELSON 


150- 


iooH 


c 
o 
o 


50^ 


o   Ouabain 
D   Propranolol 


10  3M  Ouabain  / 


-« 
10 


^  • 

Ouabain  \ 


+Propranolol 


C    10 


8 


p  Ouabain 
p  Propranolol 

FIGURE  4.  Interactive  effects  following  lO-min  incubation  in  the  beta  receptor  blocker  propranolol 
and  the  cardiac  glycoside  ouabain  on  Arbacia  sperm  motility  in  FSW.  Note  that  the  optimum  concen- 
tration of  each  drug  separately  occurs  at  10  6  A//l  FSW;  10  6  A/  is  also  the  optimum  concentration  for 
both  drugs  combined.  Also  note  that  in  the  presence  of  1  mA/  ouabain,  all  concentrations  of  propranolol 
depressed  motility.  Ordinate.  percent  of  control  progressive  motility  rate;  abscissa,  concentrations  of  the 
drugs  in  negative  log  of  molaritv. 


to  10  5  M  quinidine  after  preincubation  in  10  4  M  or  10  6  M  propranolol  (Fig.  6a) 
show  relatively  little  effect  compared  to  those  in  10~5  M  quinidine  alone. 

In  marked  contrast  sperm  cells  exposed  to  10~3M  quinidine  following  their 
preincubation  in  1CT4  M  and  10~6  M  propranolol  respectively,  first  responded  with 
motility  increases,  ranging  from  160%  to  200%  of  the  rates  in  quinidine  alone  (Fig. 
6b).  These  bursts  of  activity  were  succeeded  by  precipitous  declines  to  about  50% 
of  the  control  and  quinidine-alone  rates  on  prolonged  exposure  of  the  sperm  in  both 
cases.  In  terms  of  initial  reaction  to  addition  of  10"3  M  quinidine  to  sperm  cells  pre- 
incubated  in  propranolol,  the  response  appears  to  exceed  by  far  that  of  a  simple 
addition  of  the  individual  response  rates. 


DISCUSSION 

Membrane-stabilizing  agents  (local  anesthetics  and  antiarrhythmic  drugs)  dis- 
place calcium  from  plasma  membranes.  Perturbation  of  the  calcium  not  only  affects 
cell  permeability  (Blaustein  and  Goldman,  1966)  but  the  drugs,  as  ligands  for  Ca2+- 
binding  sites,  also  increase  contractile  tension  in  muscle  (Bondani  and  Karler,  1970). 
Displacement  of  the  calcium  required  for  biological  processes  may  enhance  or  dis- 
rupt flagellar  activity. 


CA-BLOCKERS  AND  SPERM   MOTILITY 


499 


100 


SB 


-100 


o 
o 


o 
O 


10 


p  PROPRANOLOL 


p  QUINIDINE 


FIGURE  5.  Separate  effects  of  varying  concentrations  of  propranolol  (A)  and  quinidine  (B)  on 
Arbacia  sperm  in  filtered  sea  water.  5A,  triangles,  varying  concentrations  of  propranolol  without  prein- 
cubation;  square,  effects  of  preincubating  in  propranolol  for  10  min  prior  to  rating  motility.  5B,  open 
circles,  varying  concentrations  of  quinidine  without  preincubation;  closed  circles,  same  preparation  10 
min  later,  showing  that  quinidine  does  not  alter  the  effect  of  dilution  in  FSW.  Note  that  while  higher 
concentrations  of  propranolol  markedly  depress  motile  rate,  quinidine's  effects  are  fairly  constant  over 
the  range  of  concentrations  from  O.I  nM  to  l.O  mM.  Ordinates,  relative  motility  in  percent  of  control 
rate;  abscissas,  drug  concentrations  in  negative  log  of  molar  concentration. 


Brief  exposure  to  procaine  greatly  increased  Arbacia  sperm  forward  motility; 
however,  prolonging  the  incubation  at  the  same  concentration  in  filtered  sea  water 
led  to  complete  cessation  of  progressive  movement  (Nelson,  1972).  Moreover,  EDTA 
sharply  depressed  the  swimming  rate  of  sea  urchin  sperm  (Young  and  Nelson, 
1974a).  The  calcium-selective  chelator,  EGTA,  acted  similarly  at  somewhat  lower 
concentrations  and  sharpened  the  focus  on  a  critical  role  for  Ca2+  (Young  and 
Nelson,  1974b). 

Procaine's  action  was  thought  to  reflect  an  initial  transitory  increase  in  the  in- 
ternal free  calcium  released  from  sequestration  sites  in  the  membrane;  whereas, 
blockage  of  the  cell's  ability  to  restore  calcium  to  its  resting  distribution  would 
account  for  the  delayed  inhibitory  response  (Nelson,  1972).  These  conclusions  have 
been  supported  by  the  acceleration  and  prolongation  of  motility  both  in  artificial 
sea  water  relatively  low  in  heavy  metal  contaminants,  and  following  procaine  treat- 
ment in  the  MBL-ASW.  The  motility  enhancement  in  MBL-ASW  occurs  in  contrast 
to  the  loss  of  propulsive  ability  when  the  spermatozoa  are  preincubated  in  FSW 
solutions  of  procaine.  The  immediate  response  of  the  sperm  cells  to  procaine  in 
natural  sea  water  resembles  the  responses  observed  in  synthetic  sea  water  solutions 
containing  only  minuscule  amounts  of  heavy  metals.  If  local  anesthetics  displace 
calcium  from  its  binding  sites  in  the  plasma  membrane,  then,  in  the  case  of  sea 
urchin  sperm,  at  least  part  of  that  Ca2+  which  was  released  into  the  cell  interior 
thereby  increasing  contractile  activity  was  subsequently  unable  to  be  restored  to 
physiological  levels.  In  the  synthetic  salt  medium  the  depressant  effects  of  an  excess 
of  intracellular  free  calcium  may  be  partially  alleviated  since  Ca2+  binding  sites  on 
the  outer  surface  of  the  plasma  membrane  are  not  occupied  by  heavy  metal  ligands. 


500 


LEONARD  NELSON 


6A 


QUINIDINE  10 


200  -i 

180- 

160- 

140- 

120- 

Q-10'5 

C 

P10'6 

100- 

P10'4 

p-*+Q'5 

-, 

"\ 

•n 

. 

80- 

I 

i 

; 

60- 

: 

•'. 

n 

40- 

\ 

\ 

20- 

F 

S 

W 

F 

S 

W 

F 

S 

W 

\ 

\ 

f 

s  w 

! 

O 

. 

„ 

S  10IS 


5  1015     5  1015 


5  10  IS 


5  1015 


5  10  IS 


FIGURE  6.  Interactive  effects  of  quinidine  (Q)  and  propranolol  (P)  on  progressive  movement  of 
Arbacia  sperm  in  FSW.  Fig.  6A:  In  all  panels,  sperm  cells  were  preincubated  in  FSW  for  5  min  to  establish 
control  rate  (100%).  At  5  min,  0.2  ml  of  FSW  was  added  to  the  sperm  suspensions  in  panel  C,  Q-10~5, 
P-10~4  and  P-10'6;  10  pM  quinidine  was  present  in  panels  Q-10'5,  P~4  +  Q  5  and  P~6  +  Q"5;  to  panels 
marked  P-10~4  and  P-10~6  propranolol  was  added  to  those  final  molar  concentrations.  Open  bars  con- 
tained only  the  one  drug  indicated  (quinidine  or  propranolol);  hatched  bars  represent  cuvettes  containing 
the  drug  mixtures.  Motility  ratings  were  made  at  three  5-minute  intervals.  Note  that  in  this  series  no 
significant  changes  in  the  motile  rate  were  caused  by  the  drugs.  Fig.  6B:  Similar  conditions  to  those 
depicted  in  6A,  except  that  1  mM  quinidine  was  tested  instead  of  10  ^M.  In  all  panels,  the  sperm  cells 
were  preincubated  for  5  min  in  FSW.  Then  0.2  ml  of  FSW  was  added  to  the  sperm  suspensions  in  the 
cuvettes  represented  by  the  open  bars.  To  the  hatched-bar  cuvettes  was  added  0.2  ml  of  propranolol  at 
a  final  concentration  of  10"4  M  and  10"6  M  respectively.  Panels  Q-10"3,  P~4  +  Q~3  and  P"6  +  Q'3  con- 
tained quinidine,  1  mM.  Cuvettes  represented  by  open-bar  panels  contained  only  quinidine  or  propranolol 
alone  as  indicated.  Motility  was  again  rated  at  5  min  intervals.  Note  in  this  series  that  propranolol  alone 
or  in  combination  with  quinidine  increased  the  progressive  motion  over  both  the  rates  of  the  control  and 
quinidine-alone  panels,  while  in  combination,  10~3  M  quinidine  plus  10~6  M  or  10~4  M  propranolol,  a 
marked  increase  in  sperm  speed  occurred. 


McGrady  (1979)  reported  that  10  3  Mouabain  significantly  depressed  the  mem- 
brane potential  of  bull  sperm  and  at  the  same  time  caused  decreases  in  the  frequency 
and  amplitude  of  the  flagellar  wave  as  well  as  in  progressive  movement  of  the  cells. 

Potentiation  of  these  effects  by  10~9  M  ouabain  in  MBL-ASW  suggests  that  a 
fine  and  sensitive  balance  in  the  ultimate  partition  of  the  calcium  across  the  cell 
membrane  and  within  the  cytoplasmic  components  must  be  maintained  physio- 
logically and  that  the  presence  of  heavy  metal  ions  in  the  environment  disrupts  the 
physiological  balance. 


CA-BLOCKERS  AND  SPERM   MOTILITY 


501 


QUINIDINE  10'3 


200- 

6B 

LJV" 

180- 

p;4 

160- 

t 

1 

p-6 

+ 

140- 

p 

10-4 

Q-3 

120- 

P10'6 

Q10'3 

100- 

C 

r-| 

- 

~l 

80- 

60- 

40- 

- 

I 

4 

20- 

F 

S 

W 

F 

S 

W 

F 

S 

w 

F 

S 

W 

; 

% 

y/y 

• 

; 

y/  'y 

O 

, 

^A 

FIGURE  6  (Continued} 


Ouabain  does  not  compete  for  Ca2+  binding  sites,  but  the  cardiac  glycoside 
appears  to  act  on  a  system  involved  directly  or  indirectly  in  transmembrane  Ca2+ 
extrusion.  Electron  micrographs  show  cytochemically  that,  in  Ca-loaded,  ejaculated 
bull  sperm,  ouabain  causes  the  calcium  to  accumulate  at  the  inner  surface  of  the 
plasma  membrane  (Nelson  el  al.,  1980,  1982)  as  would  be  predicted  on  the  basis 
of  inactivation  of  a  membrane-sited  calcium-extrusion  pump.  Propranolol  antag- 
onizes the  effect  of  ouabain;  the  degree  of  interaction  depends  on  the  relative  con- 
centrations of  the  drugs.  The  responses  to  the  two  drugs  may  be  ascribed  to  the 
differences  in  sites  and  modes  of  their  action  as  indicated  above.  Both  propranolol 
and  quinidine  are  cardiac  antiarrhythmics,  although  propranolol  is  a  beta-adrenergic 
receptor  blocker  and  quinidine  acts  as  a  blocker  of  alpha-adrenergic  receptors.  Quin- 
idine (Fig.  5b)  alone  caused  relatively  little  change  in  motility  from  that  of  the 
untreated  controls.  When  10~5  M  quinidine  alone  was  tested  there  was  a  40%  in- 
crease over  the  control  motility  in  FSW  (Fig.  6a),  and  when  tested  after  the  addition 
of  propranolol,  motility  remains  essentially  unaffected.  However,  when  10~4  Af  or 
10~6  M  propranolol  (final  concentration)  was  added  to  the  suspensions  preincubated 
in  10~3  M  quinidine  in  FSW,  the  motility  during  the  first  five  minutes  shot  up  to 
160%  and  200%  of  the  control  levels,  respectively,  before  dropping  back  down  to 
the  same  level  as  that  of  the  controls  during  prolongation  of  the  incubation  periods. 

Rothschild  and  Tyler  (1954)  suggested  that  sperm  cells  incubated  in  the  chem- 
ically defined  synthetic  medium  exhibit  greater  activity  for  longer  periods  by  not 


502  LEONARD  NELSON 

being  exposed  to  heavy  metal  contaminants  found  in  natural  sea  water.  However, 
the  chelating  agents  EOT  A  and  EGTA  exerted  only  depressant  effects  on  Arbacia 
sperm  motility  in  FSW  at  all  concentrations  assayed  (Young  and  Nelson,  1974b). 
Interference  with  any  of  a  number  of  Ca-dependent  processes  could  lead  to  aberrant 
behavior.  Calcium  entry  may  be  restricted  by  omission  or  removal  (e.g.  by  EGTA) 
of  calcium  from  the  cells'  environment.  Binding  sites  on  the  cell  surface  may^be 
occupied  reversibly  or  irreversibly  by  competitive  ligands  (La3+,  Cu2+,  Pb2+,  Ni2+); 
entry  channels  may  be  impeded  or  inactivated  (chelators.  La3"*,  anticholinergic 
agents);  calcium  may  be  displaced  from  sites  within  the  plasma  membrane  ("mem- 
brane-stabilizer"); binding  sites  on  Ca2+-dependent  enzymes  may  be  occupied  by 
other  cations  (Mn2+,  Zn2+);  calcium  extrusion  may  be  inhibited  (ouabain  or  inhi- 
bition of  enzymes  responsible  for  ATP  synthesis).  Sparing  ATP  by  inhibition  of 
other  ATP-utilizing  systems  also  increases  sperm  motility. 

The  time-  and  dose-dependence  of  the  responses  to  the  transmembrane  differ- 
ential distribution  of  calcium,  as  well  as  to  the  displacement  of  calcium  ions  from 
binding  sites,  appear  to  operate  in  the  case  of  the  sperm  cell  membrane  as  in  other 
excitation-effector  systems.  The  plasma  membrane  is  endowed  with  a  variety  of 
receptors  with  some  sites  showing  affinity  for  Ca2+  which  is  subject  to  displacement 
by  membrane-soluble  agents;  such  a  membrane  exhibits  selective  ionic  permeability 
and  an  environmentally  sensitive  membrane  potential. 

LITERATURE  CITED 

ANDERSON,  E.  R.,  J.  G.  FOULK.S,  AND  D.  V.  GODIN.  1981.  The  effect  of  local  anesthetics  and  antiar- 

rhythmic  agents  on  the  response  of  rabbit  platelets  to  ADP  and  thrombin.  Thromb.  Haemostasis 

45:  18-23. 
BABCOCK,  D.  F.,  J.  P.  SINGH,  AND  H.  A.  LARDY.  1979.  Alteration  of  membrane  permeability  to  calcium 

during  maturation  of  bovine  spermatozoa.  Dev.  Biol.  69:  85-93. 
BAVISTER,  B.  D.,  R.  YANAGIMACHI,  AND  R.  J.  TEICHMAN.  1976.  Capacitation  of  hamster  spermatozoa 

with  adrenal  gland  extracts.  Biol.  Reprod.  14:  219-221. 
BLAUSTEIN,  M.  P.,  AND  D.  E.  GOLDMAN.  1966.  Action  of  anionic  and  cationic  nerve-blocking  agents: 

Experiment  and  interpretation.  Science  153:  429-432. 
BONDANI,  A.,  AND  R.  KARLER.  1970.  Interaction  of  calcium  and  local  anesthetics  with  skeletal  muscle 

microsomes.  J.  Cell.  Physiol.  75:  199-212. 
COHEN,  J.  B.,  ANDJ.  P.  CHANGEUX.  1975.  The  cholinergic  receptor  protein  in  its  membrane  environment. 

Annu.  Rev.  Pharmacol.  15:  83-103. 
CORNETT,  L.  E.,  AND  S.  MEIZEL.  1978.  Stimulation  of  in  vitro  activation  and  the  acrosome  reaction  of 

hamster  spermatozoa  by  catecholamines.  Proc.  Nail.  Acad.  Sci.  75:  4954-4958. 
DUNHAM,  P.,  L.  NELSON,  AND  G.  WEISSMAN.  1982.  Effects  of  enzymatic  and  nonenzymatic  proteins 

on  Arbacia  spermatozoa:  reactivation  of  aged  sperm  and  the  induction  of  polyspermy.  Biol. 

Bull.  163:  420-430. 

GRAY,  J.  1928.  The  effect  of  dilution  on  the  activity  of  spermatozoa.  J.  E.\p.  Biol.  5:  337-344. 
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spermatozoa.  J.  Reprod.  Fertil.  56:  549-553. 
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influx.  Science  175:  1375-1377. 
NELSON,  L.  1972.  Quantitative  evaluation  of  sperm  motility  control  mechanisms.  Biol.  Reprod.  6:  319- 

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NELSON,  L.  1976.  Alpha-bungarotoxin  binding  by  cell  membranes.  Blockade  of  sperm  cell  motility.  Exp. 

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NELSON,  L.,  J.  CHAKRABORTY,  M.  YOUNG,  A.  GOODWIN,  E.  KOCK,  AND  M.  E.  GARDNER.  1980. 

Control  of  sperm  cell  motility:  neurochemical  regulation  of  calcium  transport.  Pp.  503-51 1  in 

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NELSON,  L.,  AND  A.  V.  MCGRADY.  1981.  Effects  of  ouabain  on  spermatozoan  function:  a  review.  Arch. 

Androl.  7:  169-176. 
ROTHSCHILD,  LORD.  1953.  The  movements  of  spermatozoa.  Pp.  122-133  in  Mammalian  Germ  Cells. 

G.  E.  W.  Wolstenholme,  Ed.  Little,  Brown  and  Co.,  Boston. 
ROTHSCHILD,  LORD,  AND  A.  TYLER.  1954.  The  physiology  of  sea  urchin  spermatozoa.  Action  of  versene. 

J.  Exp.  Biol.  31:  252-259. 
WOOD,  J.  M.,  C.  A.  CROW,  AND  A.  SCHWARTZ.  1972.  Ouabain-induced  respiratory  changes  in  guinea 

pig  heart  tissue.  Requirement  for  calcium.  Ada  Biol.  Med.  Germ.  28:  901-917. 
YANAGIMACHI,  R.  1970.  The  movement  of  golden  hamster  spermatozoa  before  and  after  capacitation. 

J.  Reprod.  Fertil.  23:  193-196. 
YOUNG,  L.  G.,  AND  L.  NELSON.  1974a.  The  effects  of  heavy  metal  ions  on  the  motility  of  sea  urchin 

spermatozoa.  Biol.  Bull.  147:  236-246. 

YOUNG,  L.  G.,  AND  L.  NELSON.  1974b.  Calcium  ions  and  control  of  the  motility  of  sea  urchin  sper- 
matozoa. J.  Reprod.  Fertil.  41:  371-378. 


Reference:  Biol.  Bull.  163:  504-516.  (December  1982) 


TWO  CELL  VOLUME  REGULATORY  SYSTEMS  IN  THE 

LIMULUS  MYOCARDIUM:  AN  INTERACTION  OF  IONS  AND 

QUATERNARY  AMMONIUM  COMPOUNDS 

MARY   KIM   WARREN   AND  SIDNEY   K.   PIERCE 

Department  of  Zoology.  University  of  Maryland,  College  Park,  MD  20742,  and 
Marine  Biological  Laboratory.  Woods  Hole.  MA  02543 

ABSTRACT 

The  horseshoe  crab  Limulus polyphemus  is  extremely  euryhaline.  Previous  stud- 
ies have  shown  it  surviving  in  salinities  ranging  from  6%  to  200%  sea  water.  Blood 
osmotic  concentration  is  hyperregulated  in  low  salinities,  but  above  65%  sea  water 
Limulus  is  an  osmoconformer.  Limulus  regulates  cell  volume  when  exposed  to  low 
salinity,  despite  a  small  intracellular  free  amino  acid  pool.  Instead,  the  quaternary 
ammonium  compound  glycine  betaine  is  the  major  nitrogenous  osmotic  solute  in 
Limulus  heart  tissue.  However,  volume  regulation  is  complete  before  intracellular 
glycine  betaine  concentrations  change.  Isolated  heart  tissue  exposed  to  low  salinity 
shows  no  change  in  glycine  betaine  levels  in  24  h  though  volume  regulation  occurs. 
During  the  initial  phase  of  volume  regulation  intracellular  Na+  and  Cl  content  in 
the  isolated  tissue  decreases  markedly  with  exposure  to  low  salinity.  Therefore, 
Limulus  utilizes  two  osmotic  solute  types  during  cell  volume  regulation:  Na+  and 
Cl  initially  and  glycine  betaine  later. 

INTRODUCTION 

The  utilization  of  free  amino  acids  as  intracellular  osmotic  solute  to  regulate  cell 
volume  in  euryhaline  invertebrates  exposed  to  external  osmotic  stress  has  been 
considered  ubiquitous  (Gilles,  1979;  Pierce  and  Amende,  1981).  Free  amino  acids 
often  make  up  more  than  50%  of  the  intracellular  osmotic  solute  in  marine  inver- 
tebrates (Gilles,  1979).  The  levels  of  these  intracellular  free  amino  acids  are  adjusted 
in  response  to  changes  in  extracellular  osmotic  concentration,  controlling  the  volume 
of  water  in  the  cells.  In  contrast  to  other  euryhaline  invertebrates,  the  tissues  of  the 
horseshoe  crab,  Limulus  polyphemus,  contain  only  low  levels  of  free  amino  acids, 
making  up  10%  or  less  of  the  osmotically  active  substances  (Bricteux-Gregoire  et 
al.,  1966;  Prior  and  Pierce.  1981)  despite  its  wide  salinity  tolerance.  Limulus  has 
been  found  living  in  salinities  ranging  from  7  to  30  ppt  (McManus,  1969)  and 
survived  in  experimental  salinities  of  3  to  64  ppt  (Robertson,  1970).  Furthermore, 
the  free  amino  acid  concentration  in  Limulus  tissue  drops  only  slightly  with  accli- 
mation to  low  salinity  (Prior  and  Pierce,  1981). 

Although  Limulus  cells  do  not  utilize  amino  acids  as  a  main  osmotic  solute,  the 
total  non-protein  nitrogen  content  of  Limulus  tissue  is  substantial  and  changes 
considerably  with  external  salinity  (Bricteux-Gregoire  et  al.,  1966;  Robertson,  1970). 
The  identity  of  this  nitrogenous  solute  is  unknown,  but  there  are  some  obvious 
possibilities.  In  particular,  quaternary  ammonium  compounds  are  common  in  in- 
Received  13  July  1982;  accepted  30  August  1982. 

Abbreviations:  HPLC,  high  performance  liquid  chromatography;  MOPS,  morpholinopropanesul- 
fonic  acid;  mosm,  milliosmoles  per  kilogram  water;  TCA,  trichloroacetic  acid;  TLC,  thin  layer  chro- 
matography. 

504 


LIMULUS  CELL  VOLUME  REGULATION  505 

vertebrate  tissues  (Welsh  and  Prock,  1958;  Gasteiger  et  al.,  1960;  Beers,  1967;  Rob- 
ertson, 1980)  and,  where  it  has  been  tested,  these  compounds  vary  with  salinity 
(Bricteux-Gregoire  et  al.,  1962,  1964;  Dall,  1971;  Norton  and  de  Rome,  1980)  but 
usually  constitute  only  a  minor  part  of  the  nitrogenous  osmotic  solute  pool.  Qua- 
ternary ammonium  compounds  are  present  in  Limulus  (Ackermann  and  List,  1958) 
and  might  account  for  at  least  part  of  the  unidentified  pool  of  osmotic  solute  in 
Limulus,  although  Levy  (1967)  was  unable  to  demonstrate  such  a  relationship. 

Another  possible  pool  of  intracellular  osmotic  solute  is  inorganic  ions,  although 
the  evidence  indicating  the  use  of  these  substances  by  osmotically  stressed  inver- 
tebrate cells  is  limited.  However,  inorganic  ions  play  a  role  in  volume  regulation 
in  the  few  invertebrate  species  studied,  both  as  an  initial  source  of  solute  (Kevers 
et  al.,  1981)  and  throughout  the  acclimation  period  (Freel,  1978;  Willmer,  1978). 
K+,  Na+,  and  Cl~  have  all  been  implicated  in  some  combination  (Freel,  1978;  Kevers 
et  al.,  1979;  Treherne,  1980). 

Because  of  its  extreme  euryhalinity  and  small  amino  acid  pool,  we  have  inves- 
tigated the  possible  role  of  these  alternate  solutes  in  the  cell  volume  regulation  of 
Limulus  heart  tissue.  The  results  show  that  the  quaternary  ammonium  compound 
glycine  betaine  is  the  major  nitrogenous  osmotic  solute  in  heart  tissue  taken  from 
crabs  acclimated  to  low  salinities.  However,  isolated  heart  tissue  exposed  to  low 
salinity  shows  no  decrease  in  glycine  betaine  or  K+  content  in  24  h,  but  rather  a 
large  decrease  in  Na+  and  Cl~  content.  Therefore,  Limulus  utilizes  both  organic 
compounds  and  inorganic  ions  to  regulate  cell  volume  during  low  salinity  stress. 
A  preliminary  report  of  these  findings  has  appeared  previously  (Warren  and 
Pierce,  1981). 

MATERIALS  AND  METHODS 
Limulus  acclimated  to  low  salinity 

Limulus,  obtained  from  The  Marine  Biological  Laboratory,  Woods  Hole,  MA, 
were  acclimated  to  salinities  ranging  from  930  mosm  to  55  mosm,  at  14-18°C,  for 
2-3  weeks.  Natural  sea  water,  diluted  appropriately  with  deionized  water,  was  used. 
After  the  acclimation  period,  blood  and  tissue  samples  were  taken  to  determine  the 
low-salinity  acclimated  levels  of  blood  osmotic  concentration,  tissue  amino  acids, 
total  non-protein  nitrogen  and  quaternary  ammonium  compounds,  as  indicated 
below. 

Blood  was  withdrawn  from  the  acclimated  animals  by  insertion  of  a  syringe  into 
the  pericardial  sinus  at  the  joint  between  the  prosoma  and  the  opisthosoma.  The 
blood  was  centrifuged  at  20,000  X  g  to  remove  cells  and  clots,  and  the  osmotic 
concentration  of  the  supernatant  determined  using  a  freezing  point  depression  os- 
mometer  (Precision  Systems  Osmette). 

The  heart  was  exposed  by  cutting  away  a  dorsal  section  of  the  carapace.  A  small 
section  of  the  cardiac  tissue  was  removed,  blotted,  and  weighed.  The  tissue  was  then 
lyophilized  and  reweighed  to  determine  tissue  hydration,  as  percent  wet  weight  lost 
by  lyophilization. 

Intracellular  amino  acids  were  extracted  from  the  dried  tissue  samples  by  ho- 
mogenization  in  40%  ethanol,  followed  by  boiling  and  centrifugation  to  remove 
protein.  The  supernatant  was  lyophilized  and  the  residue  resuspended  in  lithium 
citrate  buffer  (pH  2.2).  The  amino  acid  composition  of  this  solution  was  determined 
with  an  amino  acid  analyzer  (JOEL  JLC-6AH). 

Tissue  non-protein  nitrogen  concentrations  were  also  determined.  Lyophilized 
heart  tissue  was  homogenized  in  ice  cold  distilled  water.  Ice  cold  trichloroacetic  acid 


506  M.   K.   WARREN  AND  S.   K.   PIERCE 

(TCA)  was  added  to  the  homogenate  to  give  a  final  concentration  of  10%  TCA.  The 
precipitated  protein  was  spun  down  at  20,000  X  g  and  the  supernatant  frozen  until 
analysis.  A  portion  of  each  sample  was  added  to  tubes  containing  0. 1  g  digestion 
mixture  ("Sel-dahl"  copper-selenite  mixture,  Scientific  Products),  followed  by  0.5 
ml  of  concentrated  H2SO4.  The  tubes  were  heated  to  320°C  and  the  samples  digested 
at  that  temperature  for  2  h  to  break  down  nitrogenous  compounds  to  ammonia 
(Lang,  1958).  After  cooling,  the  samples  were  diluted  with  distilled  water  and  a 
portion  from  each  was  placed  into  a  glass  vial  and  neutralized  with  50%  KOH.  The 
vials  were  quickly  capped  with  rubber  stoppers,  each  of  which  held  a  glass  rod  with 
a  ground  tip  extending  into  the  vial.  Prior  to  insertion  into  the  vial,  a  drop  of  1  TV 
H2SO4  was  applied  to  the  tip  of  each  glass  rod  (Seligson  and  Seligson,  1951).  The 
vials  were  rotated  at  an  angle  overnight.  The  ammonia  released  from  the  basic 
solution  was  trapped  in  the  acid  on  the  glass  rods,  forming  ammonium  sulfate.  This 
was  rinsed  off  with  distilled  water,  and  the  ammonia  content  determined  colori- 
metrically  (Liddicoat  et  al.,  1975).  Both  ammonium  sulfate  and  glycine  betaine  were 
run  through  the  entire  procedure  as  standards. 

Intracellular  quaternary  ammonium  compounds  were  measured  in  Limulus 
cardiac  tissue  by  reineckate  precipitation  (Barnes  and  Blackstock,  1974).  Tissue 
samples  were  extracted  as  described  above  for  amino  acid  analysis,  but  the  lyoph- 
ilized  supernatants  were  resuspended  in  distilled  water  rather  than  buffer.  A  portion 
of  this  solution  from  each  tissue  sample  was  applied  to  mixed  bed  ion-exchange 
columns  (Dowex-1  and  Amberlite-50,  2:1)  and  washed  with  water,  removing  any 
interferring  amino  acids.  Then  1  N  HC1  was  added  and  mixed  with  the  washings, 
followed  by  saturated,  filtered  ammonium  reineckate  (pH  1 ).  The  quaternary  am- 
monium compounds  precipitated  while  standing  overnight  at  4°C.  The  precipitates 
were  then  filtered  from  each  solution,  using  polycarbonate  membrane  filters  (Bio- 
Rad,  0.2  yum)  in  syringe  filter  holders  (Millipore).  Once  an  entire  sample  was  filtered, 
excess  reineckate  was  removed  by  passing  ether  across  the  filter  several  times.  The 
filter  was  removed,  the  precipitate  dissolved  in  70%  acetone-water,  and  the  absor- 
bance  read  at  520  nm.  Glycine  betaine  standards  were  run  with  each  group  of 
samples. 

Preliminary  investigations  using  standard  paper  and  thin  layer  chromatography 
(TLC)  (Bregoff  ?/  al.,  1953;  Hayashi  and  Konosu,  1977)  confirmed  that  the  qua- 
ternary ammonium  compounds  glycine  betaine  and  homarine  were  present  in  Lim- 
ulus heart  tissue.  Concentrations  of  glycine  betaine  and  homarine  were  then  mea- 
sured directly  using  high  performance  liquid  chromatography  (HPLC)  (Altex).  A 
reverse  phase  column  (Spherisorb  C-6,  5  nm  particle  size,  Chromanetics)  and  a 
mobile  phase  of  0. 1  M  phosphate  buffer  (pH  3),  containing  1  mM  octane  sulfonic 
acid  as  an  ion-pairing  reagent,  were  used  to  separate  the  compounds.  Lyophilized 
samples  were  resuspended  in  water  and  a  portion  of  each,  appropriately  diluted  with 
mobile  phase  buffer,  was  injected  onto  the  column.  A  UV  monitor  (Gilson)  detected 
the  compounds  at  190  nm,  and  concentration  was  determined  with  a  data  processor 
(Shimadzu).  Identification  of  peaks  was  verified  by  spiking  samples  with  standards 
to  obtain  a  single,  larger  peak  and  by  collecting  the  column  eluent  containing  each 
peak  and  running  them  on  TLC. 

Time  course  of  acclimation 

To  determine  the  time  course  of  events  occurring  during  the  low  salinity  accli- 
mation process,  changes  in  blood  osmotic  concentration,  and  cardiac  tissue  and 
blood  glycine  betaine  levels  were  followed  from  the  time  of  transfer  of  crabs  to  low 


LIMULUS  CELL  VOLUME  REGULATION  507 

salinity  to  the  end  of  the  two-week  acclimation  period.  Prior  to  the  experimental 
period,  animals  were  maintained  at  10°C  in  artificial  sea  water  (Instant  Ocean,  930 
mosm).  Animals  were  then  transferred  from  930  mosm  to  235  mosm;  control  an- 
imals were  kept  at  930  mosm. 

Cardiac  tissue  samples  were  taken  from  both  groups  at  various  intervals  through- 
out the  two-week  acclimation  period.  Glycine  betaine  in  these  tissues  was  measured 
as  described  above.  Blood  samples  were  also  withdrawn  from  the  animals  in  both 
groups  as  previously  described.  After  centrifugation,  the  osmotic  concentration  of 
a  portion  of  each  blood  sample  was  determined.  The  remainder  of  the  blood  samples 
were  deproteinized  by  addition  of  appropriate  amounts  of  ethanol  to  a  final  con- 
centration of  40%,  brought  to  a  boil,  and  then  centrifuged  to  remove  the  precipitate. 
The  supernatant  was  analyzed  for  glycine  betaine  by  HPLC. 

Isolated  tissue  response  to  low  salinity 

The  response  of  isolated  Limulus  hearts  to  low  salinity  stress  was  also  investigated 
by  measuring  changes  in  tissue  hydration,  glycine  betaine  and  intracellular  ions.  In 
order  to  demonstrate  cell  volume  regulatory  ability  in  isolated  hearts,  initial  studies 
measured  weight  changes  in  isolated  hearts  exposed  to  low  salinity.  Hearts  were 
dissected  from  Limulus  acclimated  to  930  mosm  artificial  sea  water  (10°C)  and 
placed  in  saline  (940  mosm,  10°C,  ionic  content  in  Table  I).  The  hearts  were  carefully 
cleaned  of  any  tissue  debris  and  rinsed  several  times  with  fresh  saline.  Hearts  were 
then  put  into  either  940  mosm  or  400  mosm  saline  and  maintained  at  10°C.  (The 
400  mosm  saline  is  approximately  the  same  osmotic  concentration  as  blood  taken 
from  animals  acclimated  to  235  mosm.)  The  hearts  were  removed  from  the  saline, 
blotted,  and  weighed  at  intervals  up  to  12  or  24  h.  Changes  in  weight  were  expressed 
as  percent  initial  wet  weight  for  each  heart. 

To  measure  intracellular  ions  and  glycine  betaine,  the  dissected  hearts  were 
cleaned  and  split  longitudinally  along  the  ventral  side  and  then  cut  into  two  sections 
across  the  width.  One  section  of  each  heart  was  transferred  to  400  mosm  saline, 
while  the  other  half  was  transferred  to  940  mosm  saline,  both  solutions  containing 
14C-polyethylene  glycol  (MW  4000,  New  England  Nuclear)  as  an  extracellular 
marker  (4  h  required  for  complete  equilibration  in  the  extracellular  space).  The 
tissue  pieces  were  maintained  at  10°C  with  aeration  and  light  shaking.  Media  and 
tissue  were  sampled  at  6  and  1 2  h  intervals.  Each  tissue  sample  at  each  interval  was 

TABLE  I 
Ionic  concentrations  of  saline  used  in  isolated  Limulus  heart  experiments. 

mA/ 


NaCl 

420 

MgCl2 

30 

MgS04 

20 

KC1 

11 

CaCl2 

11 

NaHCO, 

5 

MOPS 

5 

mosm 

940 

PH 

7.5 

Lower  salinities  were  made  by  dilution  with  distilled  water,  but  maintaining  the  MOPS  concentra- 
tion. 


508  M.   K.   WARREN  AND  S.   K.   PIERCE 

divided  into  three  pieces.  Each  piece  was  blotted  and  weighed.  One  was  lyophilized 
and  reweighed  to  determine  tissue  hydration,  and  then  used  for  measurement  of 
glycine  betaine,  as  described  previously.  The  ions  were  extracted  from  a  second 
tissue  piece  with  1  N  nitric  acid.  The  third  piece  was  solubilized  in  Protosol  (New 
England  Nuclear)  and  the  14C-polyethylene  glycol  content  measured  by  liquid 
scintillation  counting.  Radioactivity  in  a  sample  of  the  media  was  also  determined, 
and  ratios  of  these  two  counts  were  used  to  determine  extracellular  space. 

Na+  and  K+  concentrations  were  measured  with  an  atomic  absorption  spectro- 
photometer  (Perkin-Elmer  Model  560).  Samples  of  the  tissue  extracts  and  the  in- 
cubation media  were  diluted  with  a  solution  containing  1%  nitric  acid  and  an  excess 
of  K+  or  Na+  as  applicable  to  prevent  ionization.  Standards  also  contained  Ca+ 
and  Mg++  in  concentrations  proportional  to  sea  water.  Chloride  was  measured  in 
samples  of  extracting  fluid  and  media  using  a  chloridometer  (Buchler-Cotlove). 

Intracellular  ion  concentrations  in  acclimated  animals 

Intracellular  ion  concentrations  were  measured  in  hearts  taken  from  animals 
acclimated  to  low  salinity  for  14  to  16  days.  Limulus  were  acclimated  to  either  930 
mosm  or  235  mosm  at  10°C.  The  acclimation  salinity  of  235  mosm  results  in  blood 
osmotic  concentrations  of  approximately  400  mosm,  the  osmotic  concentration 
used  for  the  isolated  tissue  experiments. 

After  the  acclimation  period,  blood  was  collected  from  the  animals  and  centri- 
fuged  as  previously  described.  The  hearts  were  then  removed  and  quickly  cleaned 
without  rinsing  in  saline.  Two  pieces  of  tissue  were  excised  from  each  heart  and 
quickly  processed:  one  was  blotted,  weighed,  and  lyophilized  for  determination  of 
tissue  hydration;  the  other  was  blotted,  weighed,  and  placed  in  1  A^  nitric  acid  for 
ion  extraction.  Na+,  Cl  ,  and  K+  were  measured  as  previously  described.  The  re- 
mainder of  each  heart  was  incubated  in  blood,  collected  from  the  animal,  to  which 
14C-polyethylene  glycol  was  added.  The  tissue  sections  were  maintained  with  aer- 
ation and  shaking  for  4  h,  to  allow  equilibration  of  the  polyethylene  glycol  in  the 
extracellular  space.  A  piece  of  the  heart  was  then  removed,  blotted,  and  weighed. 
The  tissue  and  a  portion  of  the  blood  were  solubilized  in  Protosol  and  radioactivity 
determined  by  liquid  scintillation  counting.  All  intracellular  ion  concentrations  were 
calculated  with  correction  for  the  extracellular  space  (Freel  et  al.,  1973). 

Statistical  analysis 

Statistical  significance  was  determined  by  analysis  of  variance  and  Student's  / 
test.  A  probability  of  P  <  0.02  was  considered  significant.  All  data  are  expressed  as 
means  ±  S.E. 

RESULTS 
Limulus  acclimated  to  low  salinities 

In  salinities  from  700  mosm  to  930  mosm,  the  blood  osmotic  concentration  of 
Limulus  varies  directly  with  that  of  the  external  medium.  Over  this  salinity  range, 
the  blood  is  slightly  hyperosmotic  to  the  medium  (16  to  27  mosm)  (Fig.  1).  In  the 
more  dilute  salinities,  from  55  mosm  to  600  mosm,  the  blood  osmotic  concentration 
is  maintained  well  above  that  of  the  medium  (52  to  307  mosm)  as  both  Robertson 
(1970)  and  Mangum  et  al.  (1976)  reported. 

Tissue  hydration  remained  constant  over  the  entire  salinity  range  tested  (Fig. 
2).  Analysis  of  variance  revealed  no  significant  differences  in  treatment  means. 


LIMULUS  CELL  VOLUME   REGULATION 


509 


lOOOr 


800 


600 


o 

E 


400 


200 


200 


400 


600 


800 


1000 


mOsm/  Kg  H  0  (external) 


FIGURE  1.    Blood  osmotic  concentration  of  Limnlus  acclimated  to  a  range  of  salinities. 

The  total  size  of  the  free  amino  acid  pool  in  heart  tissue  taken  from  Limulus 
acclimated  to  full  strength  sea  water  (930  mosm)  is  170  /zmoles/g  dry  weight.  The 
pool  size  generally  decreases  with  acclimation  to  lower  salinities  (Fig.  3).  The  major 
amino  acid  in  Limulus  heart  tissue  is  taurine  (Table  II),  making  up  nearly  50%  of 
the  total  pool  at  930  mosm  and  decreasing  with  salinity,  especially  at  the  lower 
acclimation  salinities.  In  contrast,  the  total  non-protein  nitrogen  in  cardiac  tissue 
is  nearly  1 300  )umoles/g  dry  weight  and  shows  substantial  decrease  with  acclimation 
to  low  salinity  (Fig.  4). 

A  large  portion  of  the  non-protein  nitrogen  is  accounted  for  by  quaternary 
ammonium  compounds,  which  are  750  /umoles/g  dry  weight  in  hearts  of  animals 
acclimated  to  930  mosm  (Fig.  4).  Furthermore,  the  quaternary  ammonium  com- 
pound concentration  decreases  with  acclimation  to  700  mosm  and  460  mosm  sa- 
linities. Glycine  betaine  and  homarine  account  for  most  of  the  quaternary  am- 
monium compound  pool  (514  and  139  /nmoles/g  dry  weight,  respectively)  at  930 
mosm  acclimation  salinity  (Fig.  5).  Glycine  betaine  concentration  decreases  sub- 
stantially over  the  range  of  acclimation  salinities,  whereas  homarine  shows  only  a 
slight  decrease  (Fig.  5). 


100 


80 


60 


4O 


20 


200  4OO  600  800 

External     osmotic      concentration     (mOsm) 


IOOO 


FIGURE  2.    Percent  hydration  of  heart  tissue  taken  from  Limulus  acclimated  to  a  range  of  salinities. 
Standard  errors  are  smaller  than  the  size  of  the  points. 


510 


M.   K.   WARREN   AND  S.   K.   PIERCE 


200i 


*     150 


100 


50 


Ammo  ocids 


200 


400  60O 

Salinity  ( mosm) 


800 


1000 


FIGURE  3.    Total  amino  acid  pool  in  heart  tissue  taken  from  Limnlus  acclimiated  to  a  range  of 
salinities. 


Time  course  of  acclimation 

Following  transfer  directly  from  an  external  salinity  of  930  mosm  to  235  mosm, 
the  blood  osmotic  concentration  of  Limnlus  drops  rapidly  to  within  100  mosm  of 
the  final  blood  osmotic  concentration  during  the  first  24  h,  and  levels  off  within  48 
h  (Fig.  6  top).  However,  tissue  glycine  betaine  does  not  decrease  substantially  until 
48  h,  and  then  only  slowly  declines  up  to  day  7  (Fig.  6  middle).  Blood  glycine 
betaine  concentrations  reflect  the  tissue  changes,  not  reaching  a  peak  until  48  to  72 
h  and  then  gradually  declining  through  day  14  (Fig.  6  bottom). 

Isolated  tissue  response  to  low  salinity 

Isolated  Limulus  hearts  taken  from  animals  acclimated  to  930  mosm  and  then 
exposed  directly  to  400  mosm  saline  gain  140%  of  initial  wet  weight  in  2  h  (Fig.  7). 
The  weight  then  decreases  to  123%  of  original  by  12  h  and  finally  recovers  back  to 
1 1 7%  of  initial  weight  by  24  h. 

In  spite  of  this  volume  regulation,  no  significant  change  in  tissue  glycine  betaine 
concentrations  occurred  after  6,  12,  and  24  h  of  incubation  in  low  salinity  (Table 
III).  However,  significant  decreases  in  inorganic  ion  concentrations  occurred  at  these 
sampling  intervals.  Intracellular  K+  concentration  decreases  slightly  in  the  tissues 
exposed  to  low  salinity,  but  no  more  than  can  be  accounted  for  by  cell  swelling 
(Table  IV).  The  intracellular  K+  content  (mmoles/kg  dry  weight)  does  not  decrease 
in  low  salinity.  Intracellular  Na+  and  Cl~  concentrations  decrease  drastically  during 
exposure  of  the  isolated  heart  tissues  to  400  mosm  saline  (Table  V  and  VI).  This 
decrease  is  significantly  lower  than  that  predicted  by  cellular  hydration  changes,  and 
the  Na+  and  Cl~  contents  (mmoles/kg  dry  weight)  also  decrease  significantly  in  these 
tissues  exposed  to  low  salinity. 


TABLE  II 

Major  amino  acids  (nmoles/g  dry  wt  ±  S.E.)  in  heart  tissue  taken  from  Limulus  acclimated 
to  full-strength  sea  water  (930  mosm). 


Tau 
Glu 
Pro 
Arg 
Ala 
Orn 
Asp 


79.5  ±  5.7 

24.9  ±  2.8 

23.7  ±  3.3 

21.7  ±  1.8 

7.9  ±  1.1 

4.8  ±  1.5 

3.6  ±  0.8 


LIMULUS  CELL  VOLUME   REGULATION 


511 


1  Non-protein   nitrogen 

I  Quaternary  ammonium  compounds 


1200 


1000 


-   800 


600 


400 


200 


200 


400  600 

Salinity  (mosm) 


800 


1000 


FIGURE  4.    Non-protein  nitrogen  and  quaternary  ammonium  compounds  in  heart  tissue  taken  from 
Limulus  acclimated  to  the  salinities  shown. 


Ion  concentrations  in  acclimated  animals 

The  levels  of  intracellular  inorganic  ions  in  heart  tissue  taken  from  animals 
acclimated  to  low  salinity  are  different  from  those  in  the  isolated  heart  tissue  fol- 
lowing exposure  to  low  salinity  (Table  VII).  Intracellular  Na+  and  Cl  levels  in  the 
low  salinity  acclimated  animals  are  significantly  lower  than  in  the  high  salinity 
acclimated  animals.  However,  the  Na+  and  Cl~  levels  in  the  low  salinity  acclimated 
animals  are  significantly  increased  from  the  levels  in  the  isolated  tissue  after  a  12 
h  exposure  to  low  salinity.  Furthermore,  intracellular  K+  in  the  low  salinity  accli- 
mated animals  is  significantly  decreased  from  the  levels  in  high  salinity  acclimated 
animals,  even  though  K+  content  in  the  isolated  tissues  did  not  decrease. 

DISCUSSION 

The  extreme  euryhalinity  of  Limulus  can  be  accounted  for  by  two  general  phys- 
iological processes.  First,  we  found  as  did  Robertson  (1970)  that  Limulus  is  an 


600 


500 


-       400 


300 


200 


100 


Glycine  betaine 
Homanne 


200 


400  600 

Salinity  (mosm) 


800 


1000 


FIGURE  5.   Glycine  betaine  and  homarine  concentrations,  measured  by  HPLC,  in  heart  tissue  taken 
from  Limulus  acclimated  to  a  range  of  salinities. 


512 


M.   K.   WARREN  AND  S.   K.   PIERCE 


•  930  930  mosm 

•  930  —  235  mosm 

_    1000  l 

i 

o 

i      800 

§      600 
|       400 

«  < 

—  *  *  

O 

?      200 

o 
m 

31234567                      9                                14 

Days 

eooi 


600 


<°       400 


a    200 


•  930 —  930  mosm 

•  930 — •  235  mosm 


234567 
Days 


14 


•  930 — -930  mosm 

•  930  —  235  mosm 


FIGURE  6.  Time  course  of  changes  in  blood  osmotic  concentration  (top),  tissue  glycine  betaine 
(middle)  and  blood  glycine  betaine  (bottom)  of  Limulus  acclimated  to  930  mosm  and  exposed  to  235 
mosm.  Values  for  control  animals  kept  in  930  mosm  are  also  shown. 


140 


940  —  940  mosm 
940— 400mosm 


FIGURE  7.    Time  course  of  changes  in  wet  weight,  as  %  initial  wet  weight,  of  isolated  hearts,  taken 
from  Limulus  acclimated  to  930  mosm,  and  exposed  to  400  mosm  or  940  mosm. 


LIMULUS  CELL  VOLUME   REGULATION  513 

TABLE  III 

Glycine  betaine  (nmoles/g  dry  wt  ±  S.E.)  in  isolated  heart  tissue  from  Limulus  acclimated 
to  930  mosm. 

940  mosm  400  mosm 

6  h  599  ±  24  633  ±  15 

12  h  621  ±  16  631  ±  27 

24  h  585  +  21  620  ±  21 

The  low  salinity  values  are  not  significantly  different  from  the  high  salinity  controls. 

osmoregulator  in  salinities  below  600  mosm.  Second,  Limulus  has  a  substantial 
ability  to  regulate  cell  volume.  The  basis  of  this  cellular  mechanism  is  the  utilization 
of  two  types  of  intracellular  osmotic  solutes:  small  molecular  weight  nitrogenous 
compounds  and  inorganic  ions.  Unlike  many  invertebrates,  Limulus  has  only  a 
small  intracellular  free  amino  acid  pool.  Instead,  the  quaternary  ammonium  com- 
pound glycine  betaine  is  the  major  nitrogenous  osmotic  solute  in  Limulus  heart 
tissue.  This  compound  is  a  common  constituent  in  many  invertebrates,  but  usually 
in  small  amounts  (Robertson,  1961,  1965,  1980;  Beers,  1967).  Glycine  betaine  occurs 
in  substantial  amounts  in  some  molluscs  (Mytilus,  Bricteux-Gregoire  el  ai,  1964; 
Tapes,  Norton  and  de  Rome,  1980),  in  association  with  substantial  amino 
acid  pools. 

Free  amino  acid  concentrations  in  the  cells  of  intact  euryhaline  invertebrates 
normally  fall  rapidly  during  low  salinity  stress,  often  reaching  the  final  lowered 
concentration  within  a  day  or  two  (Dall,  1975;  Bartberger  and  Pierce,  1976).  In 
contrast,  glycine  betaine  concentrations  slowly  decreased  over  7  days  in  the  heart 
tissues  of  Limulus  acclimating  to  low  salinity,  long  after  the  drop  in  blood  osmotic 
concentration  occurred.  However,  the  100-fold  increase  of  blood  glycine  betaine 
concentrations  during  the  period  of  glycine  betaine  decrease  in  the  tissues  indicates 
that  glycine  betaine  is  effluxed  intact  from  the  cells,  in  a  manner  similar  to  free 
amino  acid  utilization  by  other  species  (Pierce  and  Amende,  1981).  Thus,  glycine 
betaine  is  only  slowly  utilized  as  osmotic  solute  and  not  at  all  in  the  initial  stages 
of  salinity  acclimation  in  Limulus  heart  tissue.  This  is  confirmed  by  our  isolated 
tissue  experiments. 

The  isolated  Limulus  heart  volume  regulates  during  exposure  to  hypoosmotic 
media.  The  pattern  of  volume  regulation  by  this  tissue  is  typical  of  that  found  in 

TABLE  IV 

Intracellular  A"+  in  isolated  heart  tissue  from  Limulus  acclimated  to  930  mosm. 

mmoles/kg  H2O  mmoles/kg  dry  wt 


Salinity  940  mosm  400  mosm  Predicted*  940  mosm  400  mosm 


6 

h 

112.6  ± 

7.5 

74.7  ± 

2.9 

75.0 

±  5.2 

458 

±  29 

432  ± 

14 

12 

h 

113.0± 

5.7 

83.2  ± 

3.2 

73.6 

±4.3 

432 

±  28 

453  ± 

11 

24 

h 

114.5  ± 

5.3 

88.6  ± 

3.5 

79.3 

±  4.6 

458 

±  20 

511  ± 

13 

*  Calculated  according  to  Freel  et  al.  (1973). 

The  data  are  expressed  two  ways.  K+  concentration  (mmoles/kg  H2O)  decreases  during  low  salinity 
exposure  but  only  as  much  as  predicted  by  changes  in  tissue  hydration.  There  is  no  significant  decrease 
in  K+  content  (mmoles/kg  dry  wt),  indicating  that  K+  is  not  used  as  osmotic  solute. 


514 


M.   K.   WARREN  AND  S.   K.   PIERCE 


TABLE  V 

Intracellular  Na+  in  isolated  heart  tissue  from  Limulus  acclimated  to  930  mosm. 


mmoles/kg  H2O 


mmoles/kg  dry  wt 


Salinity 


940  mosm 


400  mosm 


Predicted" 


940  mosm 


400  mosm 


6  h 
12  h 

237 
228 

.7  ± 
.9  ± 

13.0 
17.6 

79.0  ± 
46.3  ± 

16.9 
6.3 

153.1  ± 
144.5  ± 

8.9 
10.6 

913 
905 

±  51 

±  72 

437 
273 

±  89 
±  34 

*  Calculated  according  to  Freel  el  al.  (1973). 

Na+  concentration  (mmoles/kg  H2O)  decreases  substantially  during  low  salinity  exposure,  signifi- 
cantly more  than  predicted  by  hydration  changes.  Na+  content  (mmoles/kg  dry  wt)  also  shows  a  very 
significant  decrease  during  low  salinity  stress,  indicating  that  Na+  is  used  as  osmotic  solute. 


other  cell  types:  a  rapid  swelling  followed  by  an  incomplete  recovery  (reviewed  by 
Gilles,  1979).  Cellular  volume  regulation  in  response  to  hypoosmotic  stress  is 
achieved  by  a  reduction  in  the  amount  of  intracellular  organic  osmotic  solute.  In 
most  invertebrate  cells  the  solute  reduction  is  accomplished  by  a  rapid  efflux  of 
amino  acids,  but  in  the  isolated  Limulus  heart,  glycine  betaine  levels  remained 
constant  throughout  the  24  h  exposure  to  low  salinity  even  though  volume  regulation 
was  occurring.  Thus,  the  initial  control  of  cell  volume  in  the  Limulus  tissue  must 
rely  on  an  alternate  solute  source.  Our  results  indicate  that  intracellular  Na+  and 
Cl  provide  that  function.  Intracellular  Na+  and  Cl~  contents  decrease  in  isolated 
heart  tissue  exposed  to  low  salinity,  and  the  decrease  occurs  quickly,  within  the  first 
6  h.  Therefore,  the  isolated  heart  volume  regulates  utilizing  the  high  intracellular 
Na+  and  CT  contents  as  osmotic  solute,  without  any  changes  in  the  level  of  glycine 
betaine. 

The  utilization  of  Na+  and  Cl  as  initial  osmotic  solute  explains  the  lag  time 
between  the  decline  in  blood  osmotic  concentration  and  changes  in  glycine  betaine 
in  the  cells  of  the  acclimating  whole  animal.  Na+  and  Cl~  probably  serve  as  the 
initial  osmotic  solute  during  the  first  day  or  two  of  exposure  of  the  whole  animal 
to  low  salinity,  with  the  glycine  betaine  utilization  occurring  slowly  as  the  first  week 
of  acclimation  proceeds.  In  part,  glycine  betaine  replaces  Na+  and  Cl~  as  osmotic 
solute  during  the  acclimation  process,  shown  by  the  partial  return  of  Na+  and  Cl~ 
levels  towards  original. 


TABLE  VI 

Intracellular  Cl~  in  isolated  heart  tissue  from  Limulus  acclimated  to  930  mosm. 


mmoles/kg  H2O 


mmoles/kg  dry  wt 


Salinity 


940  mosm 


400  mosm 


Predicted51 


940  mosm 


400  mosm 


6  h 

199.9  ±  16.0 

60.3  ±  8.7 

124.7  ±  9.4 

780  ±  52 

352  ±  56 

12  h 

195.8  ±  13.5 

39.5  ±  4.6 

135.8  ±  12.8 

762  ±  48 

221  ±  26 

24  h 

201.6  ±  15.3 

38.8  ±  5.1 

141.0  ±  8.3 

834  ±  76 

213  ±  24 

*  Calculated  according  to  Freel  et  al.  (1973). 

Cl  concentration  (mmoles/kg  H2O)  decreases  substantially  during  low  salinity  exposure,  signifi- 
cantly more  than  predicted  by  hydration  changes.  Cr  content  (mmoles/kg  dry  wt)  also  shows  a  very 
significant  decrease  during  low  salinity  stress,  indicating  that  Cl  ,  like  Na+,  is  used  as  osmotic  solute. 


L1MULUS  CELL  VOLUME  REGULATION  515 

TABLE  VII 

Intracellular  ion  content  (mmoles/kg  dry  wt  ±  S.E.)  of  heart  tissue  taken  from  Limulus  acclimated 
to  930  mosm  or  235  mosm. 

Acclimated  animals  Isolated  tissue* 


Salinity  940  mosm  235  mosm  940  mosm  400  mosm 


Na+ 

873 

±  47 

401  ±  62 

905  ±  72 

273  ±  34 

cr 

853 

±  57 

347  ±  55 

762  ±  48 

221  ±  26 

K+ 

443 

±  16 

361  ±  11 

432  ±  28 

453  ±  11 

*  Ion  contents  of  isolated  heart  tissue  after  a  12  h  exposure  to  940  or  400  mosm  are  included  for 
comparison. 


Our  results  show  that  the  utilization  of  glycine  betaine  as  osmotic  solute  in 
Limulus  heart  tissue  is  very  different  from  the  mechanisms  of  free  amino  acid 
regulation  in  other  euryhaline  invertebrates.  It  is  clear  that  Limulus  cells  utilize  two 
very  different  types  of  osmotic  solute.  The  solute  control  mechanisms  are  unknown 
and  we  are  currently  investigating  them.  However,  our  study  indicates  that  the 
mechanisms  controlling  each  of  the  solute  levels  are  different,  functioning  with 
separate  time  courses.  In  spite  of  this  difference,  the  mechanisms  are  coordinated 
so  that  cell  volume  is  rapidly  reduced  by  Na+  and  Cl~  efflux,  and  the  later  glycine 
betaine  efflux  continues  the  acclimation  process,  maintaining  and  perhaps  finely 
adjusting  cell  volume.  Thus,  there  seem  to  be  two  permeability  control  systems 
acting  in  concert  to  regulate  cell  volume. 

ACKNOWLEDGMENTS 

This  work  was  supported  by  N.I.H.  Grant  #  GM-23731,  the  Department  of 
Zoology,  University  of  Maryland  graduate  student  research  funds,  and  Chesapeake 
Bay  Fund.  We  thank  Jim  Calais  at  Rainin  Instrument  Co.,  Woburn,  MA,  for  his 
assistance  with  the  HPLC  separation.  This  paper  is  Contribution  No.  189  from  the 
Tallahassee,  Sopchoppy,  and  Gulf  Coast  Marine  Biological  Association,  Inc. 

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PIERCE,  S.  K.,  AND  L.  M.  AMENDE.  1981.  Control  mechanisms  of  amino  acid-mediated  cell  volume 

regulation  in  salinity-stressed  molluscs.  J.  Exp.  Zool.  215:  247-257. 
PRIOR,  D.  J.,  AND  S.  K.  PIERCE.  1981.  Adaptation  and  tolerance  of  invertebrate  nervous  systems  to 

osmotic  stress.  J.  Exp.  Zool.  214:  247-258. 
ROBERTSON,  J.  D.  1961.  Studies  on  the  chemical  composition  of  muscle  tissue.  II.  The  abdominal  flexor 

muscles  of  the  lobster  Nephrops  non-egicus  (L.).  J.  E.\p.  Biol.  38:  707-728. 
ROBERTSON,  J.  D.  1965.  Studies  on  the  chemical  composition  of  muscle  tissue.  III.  The  mantle  muscle 

of  cephalopod  molluscs.  J.  Exp.  Biol.  42:  153-175. 

ROBERTSON,  J.  D.  1970.  Osmotic  and  ionic  regulation  in  the  horseshoe  crab  Limulus  polvphemus  (Lin- 
naeus). Biol.  Bull.  138:  157-183. 
ROBERTSON,  J.  D.  1980.  Osmotic  constituents  of  some  echinoderm  muscles.  Comp.  Biochem.  Phvsiol. 

67A:  535-543. 
SELIGSON,  D.,  AND  H.  SELIGSON.  1951.  A  microdiffusion  method  for  the  determination  of  nitrogen 

liberated  as  ammonia.  J.  Lab.  Clin.  Med.  38:  324-330. 
TREHERNE,  J.  E.  1980.  Neuronal  adaptations  to  osmotic  and  ionic  stress.  Comp.  Biochem.  Phvsiol.  67B: 

455-463. 
WARREN,  M.  K.,  AND  S.  K.  PIERCE.  1981.  Osmotic  solute  in  Limulus:  Whole  animal  and  isolated  tissue 

response  to  low  salinity.  Amer.  Zool.  21:  1014. 
WELSH,  J.  H.,  AND  P.  B.  PROCK.  1958.  Quaternary  ammonium  bases  in  the  coelenterates.  Biol.  Bull. 

115:  551-561. 
WILLMER,  P.  G.  1978.  Volume  regulation  and  solute  balance  in  the  nervous  tissue  of  an  osmoconforming 

bivalve  (Mytilus  edulis).  J.  Exp.  Biol.  77:  157-179. 


INDEX 


Acid-base  regulation,  108 

A  competitive  inhibition  test  for  diagnosis  of  schis- 
tomiasis  using  monoclonal  antibodies,  393 

Actinian-asteroid  interactions,  188 

Activation  of  starfish  eggs,  465 

Adaptive  significance  of  semilunar  cycles  of  larval 
release  in  fiddler  crabs  (genus  Uca):  test  of  an 
hypothesis,  251 

ADELMAN,  W.  J.,  JR.,  A.  J.  HODGE,  AND  R.  B. 
WALTZ,  Trigonometric  nearest  neighbor  anal- 
ysis of  the  neuroplasmic  lattice  arrays  in  ax- 
ons,  379 

ADELMAN,  W.  J.,  JR.,  see  E.  F.  Stanley,  403 

A  gill  disease  of  Limulus  polyphemus  associated 
with  triclad  turbellarid  worm  infection,  392 

Aggregation  factor  complex  of  Microciona,  378, 
438 

Agglutination,  438 

AIGMEN,  SEYMOUR,  TERESA  PAXHIA,  BLENDA 
ANTONELLIS,  AND  WILLIAM  WALDRON,  Ef- 
fects of  H2O2  on  the  dogfish  (Mustelus  canis) 
ocular  lens,  404 

ALANTALO,  PHILIP,  see  Carl  J.  Berg  Jr.,  397 

ALBERTE,  R.  S.,  see  Craig  J.  Anmuth,  355,  Robert 
D.  Smith,  368,  and  W.  C.  Dennison,  364 

ALLEWELL,  NORMA,  see  George  Q.  Daley,  357 

Algal  pigments,  seasonal  variation  in  flux,  363 

ALKON,  DANIEL  L.,  see  Joseph  Farley,  383,  399 

ALLEN,  ROBERT  D.,  RAYMOND  J.  LASER,  SUSAN 
P.  GILBERT,  ALAN  J.  HODGE,  AND  C.  K. 
GOVIND,  Fast  axonal  transport  in  lobster  ax- 
ons,  379 

ALLEN,  ROBERT  D.,  see  Anthony  C.  Breuer,  381, 
and  A.  J.  Hodge,  384 

ALLIEGRO,  M.,  see  H.  Schuel,  377 

A  low  molecular  weight  subunit  of  the  aggregation 
factor  complex  of  Microciona  prolifera  that 
stoichiometrically  binds  to  and  inhibits  the 
intact  aggregation  factor,  378 

Ammocoetes,  see  lamprey 

Anatomy  and  fine  structure  of  the  eye  in  fish.  IV 
ciliary  type  tissue  in  nine  species  of  teleosts. 
The,  131 

ANDERSON,  CATHLEEN,  ABBY  M.  RICH,  ADAM 
DICKER,  PHILIP  DUNHAM,  AND  GERALD 
WEISSMANN,  Stimulus/response  coupling  in 
sponge  aggregation:  evidence  for  calcium  as 
an  intracellular  messenger,  371 

An  echinoderm  vitellaria  with  a  bilateral  larval 
skeleton:  evidence  for  the  evolution  of  ophiu- 
riod  vitellariae  from  ophioplutei,  431 

An  endopeptidase  inhibitor,  similar  to  vertebrate 
«-2  macroglobulin,  present  in  the  plasma  of 
Limulus  polyphemus,  402 

A  new  method  for  preparing  marine  eggs  for  mi- 
croinjection:  the  "fly  paper"  technique,  376 


ANMUTH,  CRAIG  J.,  S.  M.  GALLAGER,  R.  MANN, 
AND  R.  S.  ALBERTE,  Glutamate  dehydroge- 
nase  activity  in  wood-  and  mud-burrowing 
bivalve  molluscs,  355 

Anoxic  decomposition  in  marshes,  370 

Antibiotics,  resistance  to  in  enteric  bacteria,  362 

Anti-inflammatory  drug,  Indomethacin,  377 

ANTONELLIS,  BLENDA,  see  Seymour  Zigman,  404 

An  unexpectedly  steep  developmental  gradient  in 
Asterias  forbesi  embryos  induced  by  anoxia, 
373 

A  phytomastigophorean  infection  of  embryonating 
sea  hares  Aplysia  californica,  393 

Aplysia,  phytomastigophorean  infection,  393 

A  possible  role  of  protein  carbomethylase  in  fer- 
tilization and  sperm  motility,  355 

Aqueous  humor,  131 

ARANOW,  C.,  J.  COHN,  AND  W.  TROLL,  A  possible 
role  of  protein  carbomethylase  in  fertilization 
and  sperm  motility,  355 

Arbacia,  effect  of  gossypol  on  sperm  ATPase,  374 
effects  of  enzymatic  and  nonenzymatic  proteins 
on  spermatozoa,  420 

A  relatively  robust,  single-trial,  associative  learning 
in  the  opisthobranch  mollusc,  Pleurobran- 
chaea  californica,  38 1 

ARMSTRONG,  PETER  B.,  see  James  P.  Quigley,  402 

A  single  calcium-mediated  process  can  account  for 
both  rapid  and  slow  phases  of  inactivation 
exhibited  by  a  single  calcium  conductance, 
398 

Associative  learning  in  Pleurobranchaea,  381 

Asterias,  meiosis  initiation  in  oocytes,  372 
embryos  induced  by  anoxia,  373 
role  of  germinal  vesicle,  374 
coelomocyte  clumping,  438 
sperm  aster  and  pronuclear  development,  453 
oocyte  maturation,  465 

Asteroidea,  348 

A  study  of  the  heat  shock  response  in  early  embryos 
of  Spisula  solidissima,  377 

Asymmetric  claw  muscle  fiber  types,  329 

Asymmetry  in  the  olfactory  system  of  the  winter 
flounder,  Pseudopleuronectes  americanus,  389 

ATEMA,  JELLE,  see  Dana  V.  Devine,  144 

ATPase,  effect  of  gossypol  on,  374 

AUGUSTINE,  GEORGE  AND  ROGER  ECKERT,  Cal- 
cium-dependent potassium  current  in  squid 
presynaptic  nerve  terminals,  397 

AVEC-DIC  analysis  of  membranous  organelle  trans- 
port, 382 

Axonal  transport,  in  lobster,  379,  in  isolated  axo- 
plasm  of  Myxic ola  infundibulum,  381 

Axons,  analysis  of  neuroplasmic  lattice  arrays,  379 
fast  axonal  transport  in  lobster,  379 
membranous  organelle  transport  in  squid,  382 
observations  during  gluteraldehyde  fixation  in 
lobster,  384 


517 


518 


INDEX   TO  VOLUME    163 


use  and  construction  of  carbon  fiber  electrode, 

386 

Axoplasm,  fast  axonal  transport  in  Myxicola,  381 
analysis  of  membranous  organelle  transport  in 

squid,  382 


B 

Bacteria,  numbers,  diversity,  and  distribution,  366 
halophilic,  369 
germination  properties,  370 
clump-forming,  400 

BAKER,  ROBERT,  see  Stephen  M.  Highstein,  384, 

BANGS,  JAY,  STEVEN  ZEICHNER,  ROBERT  BAR- 
KER, RJCHARD  CARTER,  AND  DYANN  WIRTH, 
Stage-specific  gene  expression  in  Plasmodium 
gallinaceum,  391 

BARKER,  ROBERT,  see  Jay  Bangs,  391 

BARLOW,  ROBERT  B.,  JR.,  Seasonal  changes  in  the 
circadian  modulation  of  sensitivity  of  the 
Limulus  lateral  eye,  380 

BARLOW,  ROBERT  B.,  JR.,  see  Leslie  Eisele,  382, 
and  Leonard  Kass,  386 

BATTELLE,  B.  A.,  see  S.  C.  Lummis,  387 

BERG,  CARL  J.,  JR.  AND  PHILLIP  ALATALO,  Re- 
productive strategies  of  bivalve  mollusks  from 
deep-sea  hydrothermal  vents  and  intertidal 
sulfide-rich  environments,  397 

Biochemical  characteristics  of  macrourid  fishes  dif- 
fering in  their  depths  of  distribution,  240 

Bivalve  molluscs,  examination  of  influx  of  dis- 
solved L-alanine,  360 
reproductive  strategies,  397 

BLOCK,  BARBARA,  EUGENE  COPELAND,  AND 
FRANK  CAREY,  Fine  structure  of  tissue  warm- 
ing the  brain  and  eye  in  tuna,  356 

Blue  crab,  162 

BODZNICK,  DAVID  AND  ANNE  W.  SCHMIDT,  So- 
matotopy  within  the  medullary  electrosensory 
nucleus  of  the  skate.  Raja  erinacea,  380 

BOLDT,  J.,  see  H.  Schuel,  377 

BOLSOVER,  S.  R.  AND  J.  E.  BROWN,  Calcium  in- 
jections increase  sensitivity  in  calcium  de- 
pleted Limulus  ventral  photoreceptor  cells, 
394 

BOSWELL,  CARL  A.,  see  Debra  Rowse-Eagle,  394 

BOUTROS,  OSIRIS,  NINA  CARACO,  WILLIAM  DEN- 
NISON,  AND  IVAN  VALIELA,  Effects  of  eutro- 
phication  on  the  increase  of  chlorophyll  a  in 
phytoplankton  from  coastal  waters,  362 

BOYLE,  PAUL  J.,  see  Marianne  Walch,  403 

Brain,  tissue  warming,  in  tuna,  356 

Branchial  epithelium.  108 

BRANDHORST,  BRUCE  P.,  see  Mark  Q.  Martindale, 
374 

BRADY,  S.  T.,  see  M.  A.  Fahim,  382 

BREUER,  ANTHONY  C.,  PETER  A.  M.  EAGLES,  SU- 
SAN P.  GILBERT,  ROBERT  D.  ALLEN,  JANIS 
METUZUALS,  DAVID  F.  CLAPIN,  AND  ROGER 
D.  SLOBODA,  Fast  axonal  transport  in  isolated 
axoplasm  of  Myxicola  infundibulum,  38 1 


BROWN,  C.  R.,  see  S.  Inoue,  373 
BROWN,  J.  E.,  see  S.  R.  Bolsover,  394 
Bryozoa,  172 

BUCK,  ELISABETH,  see  John  Buck,  398 
BUCK,  JOHN,  FRANK  E.  HANSON,  ELISABETH 
BUCK,  AND  JAMES  F.  CASE,  Mechanism  and 
function  of  synchronous  flashing  in  the  firefly 
Photinus  pyralis,  398 


Calcitic  structure  of  echinoderms,  264 

Calcium-blockers,  492 

Calcium-dependent  potassium  current  in  squid 
presynaptic  nerve  terminals,  397 

Calcium  injections  increase  sensitivity  in  calcium 
depleted  Limulus  ventral  photoreceptor  cells, 
394 

Calcium,  intracellular  messenger,  371 
conductance,  398 

Callinectes,  see  blue  crab 

Capilella,  162,  regeneration  and  maturation,  366 

CAPO,  THOMAS  R.,  see  Louis  Leibovitz,  393 

CARACO,  NINA,  see  Osiris  Boutros,  362,  and  Jon- 
athan J.  Cole,  363 

Carbon  fiber  electrode:  its  construction  and  use  in 
squid  axons.  The,  386 

Can-inns,  predator-prey  relationship,  367,  vitello- 
genesis  in  hepatopancreas  and  ovaries,  375 

Cardiac  ganglion  of  Limulus,  release  and  synthesis 
of  3H-octopamine,  387 

CAREY,  FRANK,  see  Barbara  Block,  356 

CARTER,  RICHARD,  see  Jay  Bangs,  391 

CASE,  JAMES  F.,  see  John  Buck,  398 

Cassiopeia,  320 

CASTENHOLZ,  RICHARD  W.,  see  Thomas  M. 
Schmidt,  368 

CAYER,  MARILYN,  see  Gladys  Escalona  de  Motta, 
276 

Cell  adhesion,  225 

Cell  division,  inhibition  of  asymmetric  nuclear  po- 
sitioning prior  to  unequal,  373 

Cell  volume  regulation  in  Limulus,  504 

Central  nervous  system,  probes  for  staining  of,  383 

Central  organization  of  vestibular  efferent  neurons 
in  the  toad  fish,  Opsanus  tail,  384 

CHAD,  JOHN,  see  Roger  Eckert,  398 

Chaetopterus,  358 

CHAPPELL,  R.  L.,  see  C.  J.  Karwoski,  385 

Characterization  of  a  detoxifying  enzyme  from 
squid  salivary  gland  by  use  of  Soman,  DFP, 
and  manganous  ion,  401 

Characterization  of  D-xylose  and  D-glucose  trans- 
port systems  in  Spirochaeia  aurantia,  401 

Chemical  senses,  144 

Chemoreceptor  organ  function,  144,  morphology, 

162 
on  searobin  fin  rays,  responses,  390 

CHIA,  FU-SHIANG,  see  Vicki  J.  Martin,  320 

Chloride  cell,  108 

Chlorophyll  a  in  phytoplankton,  362 


INDEX  TO  VOLUME    163 


519 


CHRISTY,  JOHN  H.,  Adaptive  significance  of 
semilunar  cycles  of  larval  release  in  fiddler 
crabs  (genus  Uca):  test  of  an  hypothesis,  25 1 

Cilia,  225 

Ciliary  epithelium,  131 

Ciliary  junctions  of  scallop  gills:  The  effects  of  cy- 
tochalasins  and  concanavalin  a,  The,  225 

Circadian  clock  generates  efferent  optic  nerve  ac- 
tivity in  the  excised  Limulus  brain,  382 

Circadian  modulation  of  sensitivity  in  Limulus  lat- 
eral eye,  380 

Circadian  rhythms,  efferent  neurotransmission  in 
Limulus,  386 

CLAIBORNE,  J.  B.,  see  David  H.  Evans,  108 

CLAPIN,  DAVID  F.,  see  Anthony  C.  Breuer,  381, 
and  Janis  Metuzals,  387 

Clumping  factor,  438 

Cnidaria,  320 

Coelomic  fluid,  438 

Coelomocyte,  438 

COHEN,  L.  B.,  AND  J.  E.  FREEDMAN,  A  relatively 
robust,  single-trial,  associative  learning  in  the 
opisthobranch  mollusc,  Pleurobranchaea  cal- 
ifornica,  381 

COHEN,  L.  B.,  see  A.  Grinvald,  383,  and  H.  S. 
Orbach,  389 

COHEN,  WILLIAM  D.,  GEORGE  M.  LANGFORD, 
AND  ROGER  D.  SLOBODA,  Temperature-in- 
duced disassembly  of  isolated  marginal  bands 
and  reassembly  of  marginal  band  tubulin,  356 

COHN,  J.,  see  C.  Aranow,  355 

Colcemid  but  not  cytochalasin  inhibits  asymmetric 
nuclear  positioning  prior  to  unequal  cell  di- 
vision, 373 

Colchicine  blocks  nerve  excitation:  an  optical 
study,  386 

COLE,  JONATHAN  J.,  SUSUMU  HONJO,  AND  NINA 
M.  CARACO,  Seasonal  variation  in  the  flux  of 
algal  pigments  to  a  deep-water  site  in  the  Pan- 
ama Basin,  363 

Common  chemical  sense,  154 

Comparative  microbiology  of  metal  surfaces  in  sea 
water,  403 

Comparative  study  of  anoxic  decomposition  in  salt 
and  freshwater  marshes.  A,  370 

Comparison  of  labeled  membrane  proteins  of 
pathogenic  and  non-pathogenic  South  Amer- 
ican trypanosomes,  394 

Concanavaiin  a,  225 

Control  of  tubulin  gene  expression  during  trans- 
formation of  Lcishmania  parasites  from 
amastigote  to  promastigote  stages,  39 1 

COPELAND,  EUGENE,  see  Barbara  Block,  356 

COPELAND,  D.  EUGENE,  The  anatomy  and  fine 
structure  of  the  eye  in  fish.  IV  ciliary  type  tis- 
sue in  nine  species  of  teleosts,  131 

CORLISS,  TERESA  L.,  see  Andrew  C.  Marinucci, 
367 

Correlation  of  electron  microscopic  fine  structure 
with  videomicroscopic  observations  in  iden- 
tified lobster  axons  during  glutaraldehyde  fix- 
ation, 384 


CORSON,  D.  WESLEY  AND  ALAN  FEIN,  Nucleotide 
injection  abolishes  the  discrete  waves  evoked 
by  vanadate  in  Limulus  photoreceptors,  395 

CORSON,  D.  WESLEY,  see  Alan  Fein,  395 

Cortical  granule  breakdown  in  Asterias,  465 

Cortical  granule  exocytosis,  337 

COSTA,  J.  E.,  The  effects  of  oil  contaminated  sed- 
iments on  the  growth  of  eelgrass,  363 

Crab,  larvae,  251,  larval  release,  287,  see  Rfiith- 
ropanopeus  harrisii 

CRESWELL,  L.,  T.  OTTER,  D.  A.  LUTZ,  AND  S. 
INOUE,  Lability  of  mitotic  spindle  microtu- 
bules  during  cell  lysis,  357 

CRONIN,  T.  W.,  see  R.  B.  Forward,  287 

Crustacean  muscle,  329 

Ctenostomata,  172 

CURTIS,  N.,  see  R.  Socci,  361 

Cyanobacterial  photosynthesis,  368 

Cycle  of  larval  release  in  fiddler  crab,  251 

Cytochalasins,  225 


D 


DALEY,  GEORGE  Q.,  AND  NORMA  ALLEWELL, 
Dissociation  constants  of  dimeric  actin  cross- 
linking  proteins,  357 

DANDEK.AR,  PRAMILA,  see  Herbert  Schuel,  337 

DDT,  global  circulation  and  distribution,  365 

Deep  sea,  240 

DEL  CASTILLO,  JOSE,  see  Gladys  Escalona  de 
Motta,  276 

DELAUW,  MARIE-FRANCE,  SCOTT  LANDFEAR, 
DlANNE  MCMAHON  PRATT,  AND  DYANN 
WIRTH,  Control  of  tubulin  gene  expression 
during  transformation  of  Leishmania  para- 
sites from  amastigote  to  promastigote  stages, 
391 

DENNISON,  WILLIAM,  see  Osiris  Boutros,  362,  and 
Robert  D.  Smith,  368 

Density  effects  on  growth  and  survival  ofSalicornia 
bigelovii  and  S.  europaea,  365 

Denitrifying  bacteria  in  the  Great  Sippewissett  Salt 
Marsh:  their  numbers,  diversity,  and  distri- 
bution, 366 

DENNISON,  W.  C.  and  R.  S.  ALBERTE,  Role  of  daily 
light  period  and  production  ofZostera  marina 
L.  (eelgrass),  364 

Developmental  gradient  in  Asterias  embryos,  373 

Development  and  geomorphology  of  Great  Sip- 
pewisset  Marsh  (Falmouth,  MA):  the  Redfield 
model  revisited,  370 

Development,  Ophiuroid,  431 

DEVINE,  DANA  V.  AND  JELLE  ATEMA,  Function 
of  chemoreceptor  organs  in  spacial  orientation 
of  the  lobster,  Homarus  americanus:  differ- 
ences and  overlap,  144 

DE  WEER,  PAUL,  see  R.  F.  Rakowski,  402 

DFP,  characterization  of  enzyme,  401 

D-glucose  transport  system  in  Spirochaeta,  40 1 

DICKER,  ADAM,  see  Cathleen  Anderson,  37 1 


520 


INDEX  TO   VOLUME    163 


Dissociation  constants  of  dimeric  actin  cross-link- 
ing proteins,  357 

Dissodactylus  primitivus,  2 1 1 

Distribution,  denitrifying  bacteria,  366 

Distribution  and  ecology  of  mysids  in  Cape  Cod 
Bay,  Massachusetts,  477 

Diversity,  denitrifying  bacteria,  366 

Does  the  Schwann  cell  of  Loligo  act  as  a  potassium 
electrode?  Optical  studies  using  potentiomet- 
ric  probes,  390 

Dogfish,  see  Mmtelus 

DOWLING,  JOHN  E.,  ERIC  M.  LASATER,  AND 
HARRIS  RIPPS,  Pharmacological  properties  of 
isolated  and  cultured  horizontal  cells  of  the 
skate  retina,  382 

DUNHAM,  PHILIP,  LEONARD  NELSON,  LESLIE 
VOSSHALL,  AND  GERALD  WEISSMANN,  Ef- 
fects  of  enzymatic  and  nonenzymatic  proteins 
on  Arbacia  spermatozoa:  reactivation  of  aged 
sperm  and  the  induction  of  polyspermy,  420 

DUNHAM,  PHILIP,  see  Cathleen  Anderson,  371 

D-xylose  transport  system  in  Spirochaeta,  401 


E 

EAGLES,  PETER  A.  M.,  see  Anthony  C.  Breuer,  38 1 , 
and  Janis  Metuzals,  387 

Echinaster,  348 

Echinoderm  calcite:  a  mechanical  analysis  from 
larval  spicules,  264 

Echinodermata,  348,  431 

Echinoid  larvae,  264 

ECKBERG,  WILLIAM  R.,  The  effects  of  quercetin 
and  ionophore  A23187  on  meiosis  initiation 
in  Spisula  and  Asterias  oocytes,  372 

ECKERT,  ROGER,  DOUGLAS  EWALD,  AND  JOHN 
CHAD,  A  single  calcium-mediated  process  can 
account  for  both  rapid  and  slow  phases  of  in- 
activation  exhibited  by  a  single  calcium  con- 
ductance, 398 

ECKERT,  ROGER,  see  George  Augustine,  397 

Ecology  of  Cape  Cod  Bay  mysids,  477 

Eelgrass,  effects  of  oil  contaminated  sediments  on 

growth,  363 

role  of  daily  light  period  and  intensity  on,  364 
root-rhizome  respiration,  368 

Effect  of  gossypol  on  Arbacia  sperm  ATPase,  374 

Effect  of  habitat  structure  on  the  predator-prey  re- 
lationship between  the  green  crab,  Carcinus 
maenas,  and  the  blue  mussel,  Mvtilus  edulis, 
The,  367 

Effect  of  heat  shock  on  nuclear  RNP  structure  in 
mammalian  cells,  375 

Effect  of  methyl  a-D-glucoside  on  the  growth  of 
enteric  bacteria:  inhibition  and  escape  from 
inhibition,  403 

Effect  of  nitrogen  in  litter  and  in  ambient  water  on 
microbial  respiration  in  Spartina  decompos- 
ing in  laboratory  microcosms,  367 

Effects  of  enzymatic  and  nonenzymatic  proteins  on 
Arbacia  spermatozoa:  reactivation  of  aged 
sperm  and  the  induction  of  polyspermy,  420 


Effects  of  eutrophication  on  the  increase  of  chlo- 
rophyll a  in  phytoplankton  from  coastal  wa- 
ters, 362 

Effects  of  H2O2  on  the  dogfish  (Mustelus  canis) 
ocular  lens,  404 

Effects  of  oil  contaminated  sediments  on  the 
growth  of  eelgrass.  The,  363 

Effects  of  quercetin  and  ionophore  A23187  on 
meiosis  initiation  in  Spisula  and  Asterias  oo- 
cytes, 372 

Effects  of  sulfide  on  cyanobacterial  photosynthesis 
in  marine  microbial  mats.  The,  368 

Effect  of  temperature  and  salinity  on  larval  devel- 
opment of  sibling  species  of  Echinaster  ( Echi- 
nodermata: Asteroidea)  and  their  hybrids,  348 

Efferent  neurotransmission  of  circadian  rhythms 
in  Limulus  lateral  eye:  single  cell  studies,  386 

Efferent  optic  nerve  activity  in  excised  Limulus 
brain,  382 

Eggs,  preparing  for  microinjection,  376 

EHRLICH,  B.  E.,  A.  FINKELSTEIN,  M.  FORTE,  C. 
K.UNG,  Incorporation  of  a  calcium-selective 
conductance  from  Paramecium  cilia  in  a 
planar  lipid  bilayer,  398 

EISELE,  LESLIE,  LEONARD  KASS,  AND  ROBERT  B. 
BARLOW,  JR.,  Circadian  clock  generates  ef- 
ferent optic  nerve  activity  in  the  excised  Lim- 
ulus brain,  382 

Electrical  conduction  system  in  sea  anemone,  188 

Electrochemical,  electron  spin  resonance  and  spec- 
troscopic  measurements  of  some  cytotoxic 
quinones,  399 

Electrogenic  Na+/K+  pump  current  and  flux  mea- 
surements on  voltage-clamped,  internally  di- 
alyzed  squid  axons,  402 

Electron  microscopy,  observations  of  microbial 
colonization,  371 

Electron  spin  resonance  of  some  cytotoxic  qui- 
nones, 399 

Elongation  of  microvilli,  337 

EM  and  AVEC-DIC  analysis  of  membranous  or- 
ganelle  transport  in  squid  giant  axons  and  iso- 
lated asoplasm,  382 

Embryos,  heat  shock  response  in  Spisula,  377 

EMLET,  RICHARD  B.,  Echinoderm  calcite:  a  me- 
chanical analysis  from  larval  spicules,  264 

ENGLER,  MARLIES,  see  Jean  Hartman,  365 

Enteric  bacteria,  resistance  to  antibiotics  and  heavy 
metals,  and  the  occurrence  of  plasmids,  362 
effect  of  methyl  o-D-glucoside  on  growth,  403 

Epithelium,  ocular  lens,  360 

ESCALONA  DE  MOTTA,  GLADYS,  DAVID  S.  SMITH, 
MARILYN  CAYER,  AND  JOSE  DEL  CASTILLO, 
Mechanism  of  the  excitation-contraction  un- 
coupling of  frog  skeletal  muscle  by  form- 
amide,  276 

Escherichia,  L.  enriettii  a-tubulin  produced,  393 

Eutrophication,  effects  of  on  chlorophyll  a  in  phy- 
toplankton, 362 

EVANS,  DAVID  H.,  J.  B.  CLAIBORNE,  LINDA 
FARMER,  CHARLES  MALLERY,  AND  EDWARD 
J.  KRASNY,  JR.,  Fish  gill  ionic  transport: 
methods  and  models,  108 


INDEX  TO  VOLUME    163 


521 


EVANS,  TOM,  TIM  HUNT,  AND  JIM  YOUNGBLOM, 
On  the  role  of  maternal  mRNA  in  sea  urchins: 
studies  of  a  protein  which  appears  to  be  de- 
stroyed at  a  particular  point  during  each  cell 
division  cycle,  372 

EVERITT,  BETTY,  see  Diethardt  Jebram,  172 

Evidence  for  postnatal  morphogenesis  of  skate 
rods,  396 

Evidence  for  the  association  of  high  molecular 
weight  proteins  (MAP  2)  with  a  subset  of 
microtubules  in  vitro,  359 

Evidence  for  the  release  of  a  catalytic  agent  during 
the  latent  period  of  invertebrate  phototrans- 
duction,  396 

Evolution,  Ophiuroid,  431 

EWALD,  DOUGLAS,  see  Roger  Eckert,  398 

Excitation-contraction  uncoupling  of  frog  skeletal 
muscle,  276 

External  staining  kits  for  CNS  or  single  neuron, 
383 

Eye,  tissue  warming,  in  tuna,  356 


FAHIM,  M.  A.,  S.  T.  BRADY,  A.  HODGE,  AND 
R.  J.  LASEK,  EM  and  AVEC-DIC  analysis  of 
membranous  organelle  transport  in  squid 
giant  axons  and  isolated  axoplasm,  382 

FARLEY,  JOSEPH  AND  DANIEL  L.  ALKON,  Trans- 
duction  and  voltage-dependent  currents  of 
statocyst  hair  cells  in  Hennissenda,  399 

FARLEY,  JOSEPH,  WILLIAM  G.  RICHARDS,  LOR- 
RAINE LING,  EMILY  LIMAN,  AND  DANIEL  L. 
ALKON,  Membrane  changes  in  a  single  pho- 
toreceptor  cause  retained  associative  behav- 
ioral changes  in  Hennissenda,  383 

FARMANFARMAIAN,  A.,  see  R.  Socci,  361 

FARMER,  LINDA,  see  David  H.  Evans,  108 

Fast  axonal  transport,  in  lobster  axons,  379 
in  isolated  axoplasm  of  Myxicola,  381 

FEIN,  ALAN  AND  D.  WESLEY  CORSON,  Intracel- 
lular  injection  of  ATP  can  reduce  spontaneous 
discrete  wave  activity  in  Limulus  ventral  pho- 
toreceptors,  395 

FEIN,  ALAN,  see  D.  Wesley  Corson,  395,  and 
Richard  Payne,  396 

FENNELLY,  GLENN,  J.,  see  Janis  Metuzals,  387 

Fertilization,  envelope,  337,  355 
site  in  Hydractinia,  372 
membrane  formation  in  Asterias,  465 

Fiddler  crab,  see  Uca 

Fine  structure  of  a  scyphozoan  planula,  Cassiopeia 
xamachana,  320 

Fine  structure,  131,  of  tissue  warming  brain  and 
eye  in  tuna,  356 

FINGER,  THOMAS  E.,  see  [Catherine  Kalil,  385, 
P.  D.  Prasada  Rao,  389,  and  Wayne  L.  Silver, 
390 

FINGER,  THOMAS  E.,  Somatotopy  in  the  represen- 
tation of  the  pectoral  fin  and  free  fin  rays  in 
the  spinal  cord  of  the  searobin,  Prionotus  car- 
olinus,  154 


FiNKELSTElN,  A.,  see  B.  E.  Ehrlich,  398 

Fish  gill  ionic  transport:  methods  and  models,  108 

Fluorescent  probe,  lucifer  yellow  CH,  379 

Flux  measurements  on  squid  axons,  402 

Fly  paper  technique,  preparing  marine  eggs  for 

microinjection,  376 
Formamide,  276 

FORTE,  M.,  see  B.  E.  Ehrlich,  398 
FORWARD,  R.  B.,  JR.,  K.  LOHMANN,  AND  T.  W. 
CRONIN,  Rhythms  in  larval  release  by  an  es- 
tuarine  crab  (Rhithropanopeus  harrisii),  287 
FREEDMAN,  J.  E.,  see  L.  B.  Cohen,  381 
FREEMAN,  GARY,  The  ontogeny  of  the  fertilization 
site  in  Hydractinia  echinata  (Hydrozoa),  372 
Frequency  of  resistance  to  selected  antibiotics  and 
heavy  metals  and  the  occurrence  of  plasmids 
in  enteric  bacteria  from  a  marine  source,  362 
Freshwater  marsh,  anoxic  decomposition,  370 
Freshwater  turtles,  photoreceptors  of,  396 
FUJITA,  RODNEY  M.,  Nutrient  flux  and  growth  of 
the  red  alga  Gracilaria  tikvahiae  McLachlan 
(Rhodophyceae),  364 
Fiindulus,  semilunar  spawning  cycle,  369 
Further  studies  on  the  ultrastructure  and  distri- 
bution of  lateral  line  and  ocular-associated 
structures  (possibly  sensory)  in  a  marine  te- 
leost  (Stenotomus  chrysops),  358 


GALLAGER,  S.  M.,  see  Craig  J.  Anmuth,  355,  and 
Robert  D.  Prusch,  360 

GALLANT,  P.,  see  R.  M.  Gould,  400,  and  James 
P.  Quigley,  402 

GARDNER,  JEFF,  see  Susan  Boutros,  362 

Gene  expression,  stage-specific  in  Plasmodium, 
391 

Geomorphology  of  Great  Sippewisset  Marsh,  370 

Germination  properties  of  a  marine  spore-forming 
bacterium,  370 

Germinal  vesicle,  role  in  protein  synthesis  in  As- 
terias oocytes,  374 
breakdown  in  Asterias,  453,  465 

GILBERT,  SUSAN  P.,  see  Robert  D.  Allen,  379,  and 
Anthony  C.  Breuer,  381 

Gill,  108 

Glassworts,  see  Salicornia 

GLEESON,  RICHARD  A.,  Morphological  and  be- 
havioral identification  of  the  sensory  struc- 
tures mediating  pheromone  reception  in  the 
blue  crab,  Callinectes  sapidus,  162 

Global  circulation  and  distribution  of  DDT,  The, 
365 

Glucose  transport  mutants,  selection  and  proper- 
ties in  Vibrio,  401 

Glutamate  dehydrogenase  activity  in  wood-  and 
mud-burrowing  bivalve  molluscs,  355 

Glutaraldehyde  fixation,  in  lobster  axons,  384 

GOODE,  DENNIS,  AND  VIDYA  SARMA,  Isolation 
and  study  of  metaphase  and  anaphase  meiotic 
spindles  from  Chaetopterus  oocytes,  358 


522 


INDEX   TO   VOLUME    163 


GORDON,  DORIA  R.,  The  global  circulation  and 
distribution  of  DDT,  365 

Gossypol,  effect  on  Arbacia  sperm  ATPase,  374 

GOULD,  R.  M,  C.  A.  MANCUSO,  P.  GALLANT,  AND 
I.  TASAKJ,  Incorporation  of  32P-phosphate 
into  lipids  and  proteins  by  intact  squid  giant 
axons,  400 

GOVIND,  C.  K.,  see  Robert  D.  Allen,  379,  and 
A.  J.  Hodge,  384 

Gracilaria,  see  red  alga 

GRASSLE,  JUDITH  P.,  see  Susan  D.  Hill,  366 

Great  Sippewisset  Marsh,  370 

GREENBERG,  E.  P.,  see  N.  Wogrin.  371.  and  Cyn- 
thia A.  Paden,  401 

GREENBERG,  MICHAEL  J.,  see  Charlene  Reed- 
Miller,  225 

GRINVALD,  A.,  R.  HILDESHEIM,  J.  PINE,  AND 
L.  B.  COHEN,  Kits  of  voltage-sensitive  flu- 
orescent probes  for  external  or  iontophoretic 
staining  of  central  nervous  systems  or  single 
neurons,  383 

GRINVALD,  A.,  see  H.  S.  Orbach,  389 

GROFF,  JOSEPH  M.  AND  Louis  LEIBOVITZ,  A  gill 
disease  of  Limiilus  polyphemus  associated 
with  triclad  turbellarid  worm  infection,  392 

Growth  and  regeneration  patterns  in  the  fiddler 
crab,  Uca  pugilator,  30 1 

Growth  of  red  alga,  Gracilaria,  364 


H 


Habitat  structure,  effect  on  predator-prey  relation- 
ship, 367 

HAEDRICH,  RICHARD  L.,  see  Joseph  F.  Siebenaller, 
240 

HAIMO,  LEAH  T.,  Regions  of  microtubule  assembly 
in  isolated  spindles  ofSpisula  solidissima,  358 

HALL,  ROBERT  R.,  H.  O.  HALVORSON,  AND  K. 
KEYNAN,  Isolation  of  an  extreme  clump- 
forming  bacterium,  400 

HALVORSON,  H.  O..  see  P.  Wier,  370,  and  Robert 
R.  Hall.  400 

HAMLETT,  NANCY  V.,  see  Susan  Boutros,  362 

HANSON,  FRANK  E.,  see  John  Buck,  398 

HARDING,  CLIFFORD  V.,  see  Woo-Kuen  Lo,  360 

HARDING,  CLIFFORD  V.,  STANLEY  R.  SUSAN, 
WOO-KUEN  Lo,  S.  GREGORY  SMITH,  AND 
VINAY  REDDY,  Further  studies  on  the  ultra- 
structure  and  distribution  of  lateral  line  and 
ocular-associated  structures  (possibly  sensory) 
in  a  Marine  teleost  (Stenolomus  chrysops),  358 

HARN,  DON,  see  R.  Paul  Johnson,  392,  Martin 
Pammenter,  393,  and  Dan  Zilberstein,  394 

HARTMAN,  JEAN  M.  AND  MARLIES  ENGLER,  Den- 
sity effects  on  growth  and  survival  of  Salicor- 
nia  bigelovii  and  S.  enropaea,  365 

HARTMAN,  JEAN  M.,  see  Edwin  K.  Silverman,  368 

HASCHEMEYER,  AUDREY  E.  V.,  see  Roger  Persell, 
360 

HASCOYNE,  PETER  R.  C.,  JANE  A.  MCLAUGHLIN, 
RONALD  PETHIG,  AND  ALBERT  SZENT- 
GYORGYI,  Electrochemical,  electron  spin  res- 


onance and  spectroscopic  measurements  of 
some  cytotoxic  quinones,  399 
HAYS,  T.  S.,  see  E.  D.  Salmon,  361 
Heat  shock  response  in  embryos  of  Spisu/a,  377 

effect  on  nuclear  RNP  structure,  375 
Heavy  metals,  resistance  to  in  enteric  bacteria,  362 
HEIMBROOK,  M.  E.  AND  J.  S.  POINDEXTER,  De- 
nitrifying bacteria  in  the  Great  Sippewissett 
Salt  Marsh:  their  numbers,  diversity,  and  dis- 
tribution, 366 
HELFRICH,  JOHN  V.  K.,  see  Andrew  C.  Marinucci, 

367 

HENDLER,  GORDON,  An  echinoderm  vitellaria 
with  a  bilateral  larval  skeleton:  evidence  for 
the  evolution  of  ophiuroid  vitellariae  from 
ophioplutei,  431 

Hepatocytes,  L-leucine  transport  by,  360 
Hepatopancreas,  vitellogenesis  in  Carcinits,  375 
Hermissenda,  statocyst  hair  cells,  399 
HIGHSTEIN,  STEPHEN  M.  AND  ROBERT  BAKER, 
Central  organization  of  vestibular  efferent 
neurons  in  the  toad  fish,  Opsanus  fan,  384 
HILDESHEIM,  R.,  see  A.  Grinvald,  383 
HILL,  SUSAN  D.,  JUDITH  P.  GRASSLE,  AND  SUSAN 
W.  MILLS,  Regeneration  and  maturation  in 
two  sympatric  Capitella  (Polychaeta)  sibling 
species.  366 
Histochemistry,  329 
Ilirudo,  optical  signals  from  neurons  and  processes, 

388 

HOBBIE,  JOHN  E.,  see  Andrew  C.  Marinucci,  367 
HODGE,  A.  J.,  C.  K.  GOVIND,  R.  J.  LASEK,  AND 
R.  D.  ALLEN,  Correlation  of  electron  micro- 
scopic fine  structure  with  videomicroscopic 
observations  in  identified  lobster  axons  during 
glutaraldehyde  fixation,  384 
HODGE,  ALAN  J.,  see  Robert  D.  Allen,  379,  W.  J. 

Adelman,  379,  and  M.  A.  Fahim,  382 
Homarus,  fast  axonal  transport,  144,  379 
HONJO,  SUSUMU,  see  Jonathan  J.  Cole,  363 
HOPKINS,  PENNY  M..  Growth  and  regeneration 
patterns  in  the  fiddler  crab,  Uca  pugilator,  30 1 
Horizontal  cells  of  skate  retina,  properties,  382 
HOSKIN,  FRANCIS  C.  G.  AND  ROBERT  D.  PRUSCH, 
Characterization  of  a  detoxifying  enzyme  from 
squid  salivary  gland  by  use  of  Soman,  DFP, 
and  manganous  ion,  401 

HOWARTH,  ROBERT  W.,  see  Joanne  Willey,  370 
HUFNAGEL,  LINDA  A.,  Some  membrane  structural 
changes  accompanying  morphogenetic  changes 
in  Tetrahymena,  359 

HUMPHREYS,  TOM,  See  Pachara  Verakalasa,  378 
HUNT,  TIM,  see  Tom  Evans,  372 
Hyaline  layer,  337 
Hybrid,  348 

Hydractinia,  ontogeny  of  fertilization  site,  372 
Hydrothermal  vents,  bivalve  mollusks,  397 


Identification  of  protective  antigens  ofSchistosoma 
mansoni  by  Eastern  blots  using  monoclonal 
antibodies,  392 


INDEX  TO  VOLUME    163 


523 


Ionic  transport,  108 

lonophore  A23187,  effect  on  meiosis  initiation, 
372 

lonophoretic  staining  kits,  383 

Inactivation,  rapid  and  slow  phases,  398 

Incorporation  of  a  calcium-selective  conductance 
from  Paramecium  cilia  in  a  planar  lipid  bi- 
layer,  398 

Incorporation  of  32P-phosphate  into  lipids  and  pro- 
teins by  intact  squid  giant  axons,  400 

Indomethacin,  an  anti-inflammatory  drug,  pro- 
motes polyspermy  in  sea  urchins,  377 

Induced  maturation  by  1-methyladenine  in  Aster- 
ias, 465 

Inorganic  ions  as  osmotic  solute,  405 

INOUE,  S.,   S.   B.   POTREBIC,  C.   R.   BROWN,   AND 

D.  A.  LUTZ,  An  unexpectedly  steep  devel- 
opmental gradient  in  Asterias  forbesi  embryos 
induced  by  anoxia,  373 

INOUE,  S.,  see  L.  Creswell,  357,  Douglas  A.  Lutz, 
373,  and  R.  I.  Woodruff,  379 

Interactions  of  several  heavy  metals  with  L-leucine 
transport  in  the  intestine  of  the  toadfish,  Op- 
sanns  tan,  36 1 

Intracellular  amino  acids,  405 

Intracellular  injection  of  ATP  can  reduce  sponta- 
neous discrete  wave  activity  in  Limiilns  ven- 
tral photoreceptors,  395 

Intracellular  messenger,  calcium,  371 

Intracellular  staining  with  potentiometric  dyes:  op- 
tical signals  from  identified  leech  neurons  and 
their  processes,  388 

Invertebrate  cell  volume  control  mechanisms:  a 
coordinated  use  of  intracellular  amino  acids 
and  inorganic  ions  as  osmotic  solute,  405 

In  vitro  studies  on  the  effects  of  cell-free  coelomic 
fluid,  calcium,  and/or  magnesium  on  clump- 
ing of  coelomocytes  of  the  sea  star  Asterias 
forbesi  (Echinodermata:  Asteroidea),  438 

Iris,  131 

Isolation  of  an  extreme  clump-forming  bacterium, 
400 

Isolation  and  study  of  metaphase  and  anaphase 
meiotic  spindles  from  Chaetopterus  oocytes, 
358 


JEBRAM,  DIETHARDT  AND  BETTY  EVERITT,  New 
victorellids  (Bryozoa,  Ctenostomata)  from 
North  America:  the  use  of  parallel  cultures  in 
bryozoan  taxonomy,  172 

JOHNSON,  R.  PAUL  AND  DON  HARN,  Identification 
of  protective  antigens  ofSchistosoma  mansoni 
by  Eastern  blots  using  monoclonal  antibodies, 
392 

JOHNSON,  PAUL,  see  Martin  Pammenter,  393,  and 
Dan  Zilberstein,  394 

JUNGERY,  MICHELE,  see  Pamela  Langer,  393 

K 

KALIL,  KATHERINE,  AND  THOMAS  E.  FINGER, 
Organization  of  motoneuronal  pools  inner- 


vating muscles  of  the  free  fin  rays  in  the  sea- 
robin,  Prionotus  carolimis,  385 
KANUNGO,  K.,  In  vitro  studies  on  the  effects  of 
cell-free  coelomic  fluid,  calcium,  and/or  mag- 
nesium on  clumping  of  coelomocytes  of  the 
sea  star  Asterias  forbesi  (Echinodermata:  As- 
teroidea), 438 

KARWOSKJ,  C.  J.,  R.  L.  CHAPPELL,  L.  M.  PROENZA, 
R.  B.  SZAMIER,  D.  J.  TAATJES,  V.  MANCINI, 
AND  H.  RIPPS,  Light-evoked  field  potentials 
and  [K+]0  in  the  skate  retina:  pharmacological 
studies  on  the  cellular  origins  of  the  responses, 
385 

KASEJM,  R.  J.,  see  M.  A.  Fahim,  382 
KASS,  LEONARD,  see  Leslie  Eisele,  382 
KASS,  LEONARD  AND  ROBERT  B.  BARLOW,  JR., 
Efferent  neurotransmission  of  circadian 
rhythms  in  Limulus  lateral  eye:  single  cell 
studies,  386 

KEYNANA,  A.,  see  P.  Wier,  370 
KEYNAN,  K.,  see  Robert  R.  Hall,  400 
Kits  of  voltage-sensitive  fluorescent  probes  for  ex- 
ternal or  iontophoretic  staining  of  central  ner- 
vous system  or  single  neurons,  383 
KOIDE,  S.  S.,  see  Hideo  Mohri,  374,  and  Eimei 

Sato,  376 

KORNBERG,  H.  L.,  see  D.  J.  Schnell,  403 
KORNBERG,  H.  L.,  T.  M.  PERNACK,  AND  D.  J. 
SCHNELL,  Selection  and  properties  of  glucose 
transport  mutants  of  Vibrio  parahaemolyti- 
cus,  401 

KRASNY,  EDWARD  J.,  see  David  H.  Evans,  108 
KUNG,  C.,  see  B.  E.  Ehrlich,  398 


Lability  of  mitotic  spindle  microtubules  during  cell 
lysis,  357 

L-alanine,  uptake  and  utilization  of,  360 

Lamprey,  197 

LANDFEAR,  SCOTT,  see  Marie-France  Delauw,  391 

LANDOWNE,  DAVID,  JAMES  LARSEN,  AND  KEVIN 
TAYLOR,  Colchicine  blocks  nerve  excitation: 
an  optical  study,  386 

LANDOWNE,  DAVID,  see  James  B.  Larsen,  386 

LANGER,  PAMELA,  MICHELE  JUNGERY,  AND 
DYANN  WIRTH,  L.  enriettii  a-tubulin  is  pro- 
duced in  vivo  by  Escherichia  coli,  393 

LANGFORD,  GEORGE  M.,  AND  ADRIAN  C.  LAW- 
RENCE, Evidence  for  the  association  of  high 
molecular  weight  proteins  (MAP  2)  with  a 
subset  of  microtubules  in  vitro,  359 

LANGFORD,  GEORGE  M.,  see  William  D.  Cohen, 
356 

LARSEN,  JAMES  B.,  AND  DAVID  LANDOWNE,  The 
carbon  fiber  electrode:  its  construction  and 
use  in  squid  axons,  386 

LARSEN,  JAMES,  see  David  Landowne,  386 

Larval  development,  348 

LASATER,  ERIC  M.,  see  John  M.  Dowling,  382 

LASEK,  RAYMOND  J.,  see  Robert  D.  Allen,  379, 
and  A.  J.  Hodge,  384 

Lateral  eye,  of  Limulus,  380 


524 


INDEX   TO  VOLUME    163 


Lateral  line  distribution  in  Stenotomus,  358 
LAUFER,  HANS,  see  Jeanne  E.  Paulus,  375 
LAWN,  I.  D.  AND  D.  M.  Ross,  The  release  of  the 
pedal  disk  in  an  undescribed  species  of  Tealia 
(Anthozoa:  Actiniaria),  188 
LAWRENCE,  ADRIAN  C.,  see  George  M.  Langford, 

359 
LEFEROVICH,  JOHN  M.,  see  Kathleen  O'Connor, 

329 

LEIBOVITZ,  Louis  AND  THOMAS  R.  CAPO,  A  phy- 
tomastigophorean  infection  of  embryonating 
sea  hares  Aplysia  californica,  393 
LEIBOVITZ,  Louis,  see  Joseph  M.  Groff,  392 
Leishmania,  control  of  gene  expression  during 
transformation  from  amastigote  to  promasti- 
gote,  391 
L.  enriettii  «-tubulin  is  produced  in  vivo  by  Esch- 

erichia  coli,  393 

Light-evoked  field  potentials  and  [K+]0  in  the  skate 
retina:  pharmacological  studies  on  the  cellular 
origins  of  the  responses,  385 
LIMAN,  EMILY,  see  Joseph  Farley,  383 
Limulns,  circadian  modulation  of  sensitivity  of  the 

lateral  eye,  380 

circadian  clock  generates  activity  in  brain,  382 
circadian  rhythms  in  lateral  eye,  386 
synthesis  and  release  of  3H-octopamine,  387 
gill  disease  associated  with  turbellarid  worm  in- 
fection, 392 
increased  sensitivity   in   ventral   photoreceptor 

cells,  394 

discrete  waves  abolished  in  photoreceptors,  395 
endopeptidase  inhibitor  present  in  plasma,  402 
cell  volume  regulatory  systems,  504 
LING,  LORRAINE,  see  Joseph  Farley,  383 
LIPETZ,  LEO  E.  AND  EDWARD  F.  MACNICHOL,  JR., 
Photoreceptors  of  fresh  water  turtles:  cell  types 
and  visual  pigments,  396 
Lipids,  incorporation  of  32P-phosphate,  400 
L-leucine  transport  by  isolated  toadfish  hepato- 

cytes,  360 
L-leucine  transport,  interactions  of  heavy  metals  in 

intestine  of  Opsanus,  361 
Lobster,  see  Homarus 
LOHMANN,  K.,  see  R.  B.  Forward,  287 
Loligo,  Schwann  cell,  390 

LONGO,  FRANK  J.  AND  ALLEN  W.  SCHUETZ,  Male 
pronuclear  development  in  starfish  oocytes 
treated  with  1-methyladenine,  453 
LONGO,  FRANK  J.,  FREDERICK  So,  AND  ALLEN  W. 
SCHUETZ,  Meiotic  maturation  and  the  cortical 
granule  reaction  in  starfish  eggs,  465 
Lo,  WOO-KUEN,  AND  CLIFFORD  V.  HARDING, 
Zonulae  occludentes  and  transepithelial  per- 
meability in  the  ocular  lens  epithelium,  360 
Lo,  Woo-KUEN,  see  Clifford  V.  Harding,  358 
Lucifer  yellow  CH  as  a  non-intrusive,  in  vivo  flu- 
orescent probe  for  physiological  studies  during 
early  development,  379 

LUMMIS,  S.  C.,  P.  M.  O'CONNOR,  AND  B.  A.  BAT- 

TELLE,  Synthesis  and  release  of  3H-octopa- 


mine  from  the  cardiac  ganglion  of  Limulus 
polyphemus,  387 

LUTZ,  DOUGLAS  A.,  AND  SHINYA  INOUE,  Col- 
cemid  but  not  cytochalasin  inhibits  asym- 
metric nuclear  positioning  prior  to  unequal 
cell  division,  373 

LUTZ,  D.  A.,  see  L.  Creswell,  357,  S.  Inoue,  373, 
and  R.  1.  Woodruff,  379 

M 

MACAGNO,  EDUARDO  R.,  see  Michele  Masacchio, 
388 

MACNICHOL,  EDWARD  F.  JR.,  see  Leo  E.  Lipetz, 
396 

Macrourid  fishes,  240 

Male  pronuclear  development  in  starfish  oocytes 
treated  with  1-methyladenine,  453 

MALLATT,  JON,  Pumping  rates  and  particle  reten- 
tion efficiencies  of  the  larval  lamprey,  an  un- 
usual suspension  feeder,  197 

MALLERY,  CHARLES,  see  David  H.  Evans,  108 

Mammalian  cells,  effect  of  heat  shock  on  nuclear 
RNP  structure,  375 

MANCINI,  V.,  see  C.  J.  Kanvoski,  385 

MANCUSO,  C.  A.,  see  R.  M.  Gould,  400 

Manganous  ion,  characterization  of  enzyme,  401 

MANN,  R.,  see  Craig  J.  Anmuth,  355,  and  Robert 
D.  Prusch,  360 

Marginal  bands,  temperature-induced  disassembly 
of,  356 

Marine  rat,  monitoring  of  activity  in  visual  cortex, 
389 

MARINUCCI,  ANDREW  C.,  JOHN  E.  HOBBIE,  TE- 
RESA L.  CORLISS,  AND  JOHN  V.  K.  HELFRICH, 
Effect  of  nitrogen  in  litter  and  in  ambient 
water  on  microbial  respiration  in  Spartina 
decomposing  in  laboratory  microcosms,  367 

MARSH,  ADAM  G.,  see  Stephen  A.  Watts,  348 

MARTIN,  VICKI  J.  AND  FU-SHIANG  CHIA,  Fine 
structure  of  a  scyphozoan  planula,  Cassiopeia 
\amachana,  320 

MARTINDALE,  MARK  Q.  AND  BRUCE  P.  BRAND- 
HORST,  The  role  of  the  germinal  vesicle  in  the 
!-methyladenine-induced  changes  in  protein 
synthesis  in  Asterias  oocytes,  374 

MARZOLF,  ERICH  R.,  Potential  nitrification  rates 
in  a  salt  marsh,  367 

MASACCHIO,  MICHELE  AND  EDUARDO  R.  MA- 
CAGNO, Quantitative  aspects  of  growth  of  an 
identified  neuron  in  the  leech  Hirudo  medi- 
cinalis,  388 

MATSUDA,  KYOKO,  see  Hideo  Mohri,  374 

MAURER,  DON  AND  ROLAND  L.  WIGLEY,  Distri- 
bution and  ecology  of  mysids  in  Cape  Cod 
Bay,  Massachusetts,  477 

McCLiNTOCK,  JAMES  B.,  see  Stephen  A.  Watts, 
348 

McKEEL,  M.,  see  E.  D.  Salmon,  361 

MCLAUGHLIN,  JANE  A.,  see  Peter  R.  C.  Hascoyne, 
399 


INDEX   TO   VOLUME    163 


525 


Mechanism  and  function  of  synchronous  flashing 

in  the  firefly  Photinus  pyralis,  398 
Mechanism  of  the  excitation-contraction  uncou- 
pling of  frog  skeletal  muscle  by  formamide, 
276 
Medullary  electrosensory  nucleus,  somatotopy 

within,  380 

Meiosis  in  Asterias,  465 

Meiotic  maturation  and  the  cortical  granule  reac- 
tion in  starfish  eggs,  465 

Meiotic  spindles,  metaphase  and  anaphase,  358 
Membrane  changes  in  a  single  photoreceptor  cause 
retained  associative  behavioral  changes  in 
Hermissenda,  383 
Membrane  labeling  of  protective  antigens  of  schis- 

tosomula  of  Schistosoma  mansoni,  394 
Membrane-stabilizing  and  calcium-blocking  agents 

affect  Arbacia  sperm  motility,  492 
Membrane  structural  changes  in  Tetrahymena,  359 
Membranous  organelle  transport  in  squid  axons 

and  axoplasm,  analysis  of,  382 
MERLINO,  GLENN,  see  Kristi  Wharton,  378 
Metal  surfaces,  comparative  microbiology  in  sea 

water,  403 

METUZALS,  JANIS,  DAVID  F.  CLAPIN,  GLENN 
J.  FENNELLY,  AND  PETER  A.  M.  EAGLES, 
Paracrystalline  arrays  of  neurofilament  pro- 
tein, 387 

METUZUALS,  JANIS,  see  Anthony  C.  Breuer,  381 
Microbial  colonization  of  filter  paper  incubated  in 
saltmarsh  sediments  as  observed  by  scanning 
electron  microscopy,  371 
Microbial  respiration  in  Spartina,  367 
Microbiology  of  metal  surfaces  in  sea  water,  403 
Microciona,  aggregation  factor  complex  of,  378 
Microinjection,  preparing  marine  eggs  for,  376 
Microtubule,  lability  of  mitotic  spindle,  357 
regions  of  assembly,  358 
reconstituted  in  vitro,  359 
colchicine-  or  colcemid-induced  spindle  disas- 
sembly, 361 

implications  for  the  mechanism  of  assembly,  36 1 
MILLS,  SUSAN  W.,  see  Susan  D.  Hill,  366 
MITCHELL,  RALPH,  see  Marianne  Walch,  403 
MOHRI,  HIDEO,  KYOKO  MATSUDA,  S.  S.  KOIDE, 
AND  SHELDON  J.  SEGAL,  Effect  of  gossypol  on 
Arbacia  sperm  ATPase,  374 
Molluscs,  glutamate  dehydrogenase  activity,  355, 

see  Pleurobranchaea 
Monoclonal  antibodies,  392,  393 

MORGANELLI,  CHRISTINE  MAUTE,  Effect  of  heat 

shock  on  nuclear  RNP  structure  in  mam- 
malian cells,  375 

Morphogenetic  changes  in  Tetrahymena,  359 

Morphological  and  behavioral  identification  of  the 
sensory  structures  mediating  pheromone  re- 
ception in  the  blue  crab,  Callinectes  sapidus, 
162 

Motoneural  pools  innervating  muscles  in  searobin, 
organization  of,  385 

Muscle  fiber  types  in  Alpheus,  329 


Mustelus,  effect  of  H2O2  on  ocular  lens,  404 
Mysids  of  Cape  Cod  Bay,  477 
Mytilns,  predator-prey  relationship,  367 
Myxicola,  fast  axonal  transport  in  isolated  axo- 
plasm of,  381 

N 

NELSON,  LEONARD,  Membrane-stabilizing  and 

calcium-blocking  agents  affect  Arbacia  sperm 

motility,  492 

NELSON,  LEONARD,  see  Philip  Dunham,  420 
Neurofilament  protein,  paracrystalline  arrays,  387 
Neuron,  quantitative  aspects  of  growth  in  Hirudo, 

388 

Neurons,  probes  for  staining  of,  383 
Neuroplasmic  lattice  arrays  in  axons,  analysis  of, 

379 
New  victorellids  (Bryozoa,  Ctenostomata)  from 

North  America:  The  use  of  parallel  cultures 

in  bryozoan  taxonomy,  172 
Nitrification  rates  in  salt  marsh,  367 
Nitrogen,  effect  of  on  respiration  in  Spartina,  367 
Nuclear   positioning,   colcemid   inhibits   asym- 
metric, 373 
Nuclear  RNP  structure,  effect  of  heat  shock  on, 

375 
Nucleotide  injection  abolishes  the  discrete  waves 

evoked  by  vanadate  in  Limulm  photorecep- 

tors,  395 

Numbers,  denitrifying  bacteria,  366 
Nutrient  flux  and  growth  of  the  red  alga  Gracilaria 

tikvahiae  McLachlan  (Rhodophyceae),  364 


o 

OBAID,  A.  L.,  H.  SHIMIZU,  AND  B.  M.  SALZBERG, 
Intracellular  staining  with  potentiometric  dyes: 
optical  signals  from  identified  leech  neurons 
and  their  processes,  388 
OBAID,  A.  L.,  see  B.  M.  Salzberg,  390 
O'CONNOR,  KATHLEEN,  PHILIP  J.  STEPHENS,  AND 
JOHN  M.  LEFEROVICH,  Regional  distribution 
of  muscle  fiber  types  in  the  asymmetric  claws 
of  Californian  snapping  shrimp,  329 
O'CONNOR,  P.  M.,  see  S.  C.  Lummis,  387 
Ocular-associated  structures  in  Stenotomus,  358 
Ocular  lens  of  Mustelus,  effects  of  H2O2,  404 
Olfactory  system  of  flounder,  asymmetry,  389 
O'MELIA,  ANNE  F.,  Synthesis  of  5S  RNA  and 
tRNA  in  cleaving  sea  urchin  embryos:  effect 
of  altering  cell  interactions,  375 
1-methyladenine,  male  pronuclear  development  in 

Asterias,  453 

On  the  role  of  maternal  mRNA  in  sea  urchins: 
studies  of  a  protein  which  appears  to  be  de- 
stroyed at  a  particular  point  during  each  cell 
division  cycle,  372 

Ontogeny  of  the  fertilization  site  in  Hydractinia 
echinata  (Hydrozoa),  372 


526 


INDEX  TO   VOLUME    163 


Oocytes,  study  of  meiotic  spindles,  358 

effect  of  quercetin  and  ionophore  A23187  on, 

372 
sperm  agglutinating  factor  isolated  from  Spisula, 

376 

ultrastructure  in  Asterias,  465 
Ophiopluteus,  43 1 
Ophiuroid,  431 
Opsanus,  central  organization  of  vestibular  efferent 

neurons,  384 
Optical  monitoring  of  evoked  activity  in  the  visual 

cortex  of  the  marine  rat,  389 
Optic  nerve,  circadian  clock  generates  activity,  382 
ORBACH,  H.  S.,  L.  B.  COHEN,  AND  A.  GRINVALD, 

Optical  monitoring  of  evoked  activity  in  the 

visual  cortex  of  the  marine  rat,  389 
Organization   of  motoneural   pools   innervating 

muscles  of  the  free  fin  ray  in  the  searobin, 

Prionotus  carolinus,  385 
Orientation,  144 

ORKAND,  R.  K.,  see  B.  M.  Salzberg,  390 
Osmotic  solute,  coordinated  use  of  amino  acids  and 

ions,  405 
OTTER,  T.,  see  L.  Creswell,  357,  and  Cynthia  L. 

Sundell,  362 
Ouabain,  492 
Ovaries,  vitellogenesis  in  Carcinus,  375 


PADEN,  CYNTHIA  A.,  SUSAN  ROBERTS,  AND  E.  P. 
GREENBERG,  Characterization  of  o-xylose 
and  D-glucose  transport  systems  in  Spiro- 
chaeta  aurantia,  401 

PAMMENTER,  MARTIN,  PAUL  JOHNSON,  AND  DON 
HARN,  A  competitive  inhibition  test  for  di- 
agnosis of  schistomiasis  using  monoclonal  an- 
tibodies, 393 

Paracrystalline  arrays  of  neuronlament  protein, 
387 

Paramecium,  calcium  conductance  from  cilia,  398 

Parthenogenetic  activation,  337 

Particle  retention  efficiencies  of  larval  lamprey,  197 

PAULUS,  JEANNE  E.,  AND  HANS  LAUFER,  Vitel- 
logenesis in  the  hepatopancreas  and  ovaries 
of  Carcinus  maenas,  375 

PAYNE,  RICHARD  AND  ALAN  FEIN,  Evidence  for 
the  release  of  a  catalytic  agent  during  the  latent 
period  of  invertebrate  phototransduction,  396 

Pectoral  fin,  1 54 

Pedal  disc  release  in  sea  anemone,  1 88 

PEREIRA,  MIERCIO,  see  Tecia  Maria  Ulisses  de 
Carvalho,  39 1 ,  Dan  Zilberstein,  394,  and  De- 
bra  Rowse-Eagle,  394 

Perfusion  of  the  squid  stellate  ganglion  through  its 
blood  supply:  implications  for  morphological 
and  physiological  studies  of  the  squid  giant 
synapse,  403 

PERNACK,  T.  M.,  see  H.  L.  Kornberg,  401,  and 
D.  J.  Schnell,  403 

PERSELL,  ROGER,  AND  AUDREY  E.  V.  HASCHE- 
MEYER,  L-leucine  transport  by  isolated  toad- 
fish  hepatocytes,  360 


PETHIG,  RONALD,  see  Peter  R.  C.  Hascoyne,  399 

Pharmacological  properties  of  isolated  and  cul- 
tured horizontal  cells  of  the  skate  retina,  382 

PHAXHIA,  TERESA,  see  Seymour  Zigman,  404 

Pheromone  reception,  162 

Photoreceptor  cells,  increased  sensitivity  in  Lim- 
ii/us,  394 

Photoreceptor,  membrane  changes  causing  re- 
tained associative  behavioral  changes  in  Her- 
missenda,  383 

discrete  wave  activity  in  Limulus,  395 
freshwater  turtles,  396 

Photosynthesis,  in  eelgrass  (Zostera),  364,  shoot, 
368 

Photinus,  synchronous  flashing,  398 

Phototransduction,  release  of  catalytic  agent  during 
invertebrate,  396 

PIERCE,  SYDNEY  K.,  Invertebrate  cell  volume  con- 
trol mechanisms:  a  coordinated  use  of  intra- 
cellular  amino  acids  and  inorganic  ions  as 
osmotic  solute,  405 

PIERCE,  SIDNEY  K.,  see  Mary  Kim  Warren,  504 

PINE,  J.,  see  A.  Grinvald,  383 

Planar  lipid  bilayer,  incorporation  of  calcium  con- 
ductance, 398 

Planula,  320 

Plasma,  endopeptidase  inhibitor  found  in  Limulus, 
402 

Plasmids,  occurrence  in  enteric  bacteria,  362 

Plasmodium,  stage-specific  gene  expression,  39 1 

Plewobranchaea,  associative  learning,  381 

POCHAPIN,  MARK  BENNETT,  JEAN  M.  SANGER, 
AND  JOSEPH  W.  SANGER,  A  new  method  for 
preparing  marine  eggs  for  microinjection:  the 
"fly  paper"  technique,  376 

POHLE,  GERHARD  AND  MALCOLM  TELFORD,  Post- 
larval  growth  of  Dissodactylus  primitivusbou- 
vier,  1917  (Brachyura:  Pinnotheridae)  under 
laboratory  conditions,  2 1 1 

POINDEXTER,  J.  S.,  see  M.  E.  Heimbrook,  366,  and 
N.  Wogrin,  371 

Polychaete,  see  Capitella 

Post-larval  growth  of  Dissodactylus primitivus  bou- 
vier,  1917  (Brachyura:  Pinnotheridae)  under 
laboratory  conditions,  2 1  1 

Postnatal  morphogenesis  of  skate  rods,  396 

Potential  nitrification  rates  in  a  salt  marsh,  367 

Potentiometric  dyes,  intracellular  staining  record- 
ing optical  signals  in  Hirudo,  388 

POTREBIC,  S.  B.,  see  S.  Inoue,  373 

PRATT,  DIANNE  McMAHON,  see  Marie-France 
Delauw,  391 

Predator-prey  relationship,  effect  of  habitat  struc- 
ture, 367 

Presynaptic  nerve  terminals  in  squid,  397 

Prionotus,  154 

organization  of  motoneural  pools  innervating 

muscles  of  the  free  fin  ray,  385 
fin  ray  chemoreceptor  responses,  390 

Procaine,  492 

Production,  in  eelgrass,  (Zostera),  364 

PROENZA,  L.  M.,  see  C.  J.  Karwoski,  385 

Pronuclear  development  in  Asterias,  453 

Propranolol,  492 


INDEX  TO  VOLUME    163 


527 


Protein,  carbomethylase,  355 
dimeric  actin  cross-linking,  357 
high  molecular  weight  (MAP  2),  359 
destroyed  during  cell  division  cycle,  372 
synthesis  in  Asterias  oocytes,  374 
incorporation  of  32P-phosphate,  400 
activation  of  Arbacia  sperm,  420 
PRUSCH,  ROBERT  D.,  SCOTT  M.  GALLAGER,  AND 
ROGER  MANN,  Uptake  and  utilization  of  L- 
alanine  by  10  species  of  bivalve  molluscs,  360 
PRUSCH,  ROBERT  D.,  see  Francis  C.  G.  Hoskin, 

401 

Pseudopleuronectes,  olfactory  asymmetry,  389 
Pumping  rates  and  particle  retention  efficiencies  of 
the  larval  lamprey,  an  unusual  suspension 
feeder,  197 


Quaternary  ammonium  compounds,  504 
Quantitative  aspects  of  growth  of  an  identified  neu- 
ron in  the  leech  Hirudo  medicinal! s,  388 
Quercetin,  effect  on  meiosis  initiation,  372 
QUIGLEY,  JAMES  P.,  PETER  B.  ARMSTRONG,  PAUL 
GALLANT,  FRED  R.  RICKLES,  AND  WALTER 
TROLL,  An  endopeptidase  inhibitor,  similar 
to  vertebrate  «-2  macroglobulin,  present  in 
the  plasma  of  Limulus  polyphemus,  402 
Quinidine,  492 


R 

RAFF,  RUDOLF,  see  Kristi  Wharton,  378 

Raja,  somatotopy  within  the  medullary  electrosen- 
sory  nucleus,  380 

RAK.OWSK.I,  R.  F.  AND  PAUL  DE  WEER,  Electro- 
genie  Na+/K+  pump  current  and  flux  mea- 
surements on  voltage-clamped,  internally  di- 
alyzed  squid  axons,  402 

RAO,  P.  D.  PRASADA,  THOMAS  E.  FINGER,  AND 
WAYNE  L.  SILVER,  Asymmetry  in  the  olfac- 
tory system  of  the  winter  flounder,  Pseudo- 
pleuronectes  americanus,  389 

Rapid  rates  of  colchicine-  or  colcemid-induced 
spindle  microtubule  disassembly  in  vivo:  im- 
plications for  the  mechanism  of  microtubule 
assembly,  361 

Red  alga,  nutrient  flux  and  growth,  364 

REDDY,  VINAY,  see  Clifford  V.  Smith,  358 

Redfield  model,  revisited,  370 

REED-MILLER,  CHARLENE  AND  MICHAEL  J. 
GREENBERG,  The  ciliary  junctions  of  scallop 
gills:  The  effects  of  cytochalasins  and  con- 
canavalin  a,  225 

Regeneration  and  maturation  in  two  sympatric 
Capitella  (Polychaeta)  sibling  species,  366 

Regeneration  of  Uca  pugilator,  30 1 

Regional  distribution  of  muscle  fiber  types  in  the 
asymmetric  claws  of  Californian  snapping 
shrimp,  329 

Regions  of  microtubule  assembly  in  isolated  spin- 
dles of  Spisula  solidissima,  358 


Release  of  the  pedal  disk  in  an  undescribed  species 
of  Tealia  (Anthozoa:  Actiniaria),  The,  188 

Reproduction,  ophiuroid,  431 

Reproductive  strategies  of  bivalve  mollusks  from 
deep-sea  hydrothermal  vents  and  intertidal 
sulfide-rich  environments,  397 

Responses  from  spinally  innervated  chemorecep- 
tors  on  the  fin  rays  of  the  searobin,  Prionotus 
carolinus,  390 

Retained  associative  behavioral  changes  in  Her- 
missenda,  383 

Retina,  properties  in  skate,  382,  light-evoked  field 
potential  in  skate,  385 

REVELAS,  EUGENE  C.,  The  effect  of  habitat  struc- 
ture on  the  predator-prey  relationship  be- 
tween the  green  crab,  Carcinus  maenas,  and 
the  blue  mussel,  Mytilus  edulis,  367 

REZNIKOFF,  WILLIAM,  see  Susan  Boutros,  362 

Rhithropanopeus  harrissii,  287 

Rhythms  in  larval  release  by  an  estuarine  crab 
(Rhithropanopeus  harrissii),  287 

RICH,  ABBY  M.,  see  Cathleen  Anderson,  371 

RICHARDS,  WILLIAM  G.,  see  Joseph  Farley,  383 

RICKLES,  FRED  R.,  see  James  P.  Quigley,  402 

RIEDER,  C,  see  E.  D.  Salmon,  361 

RIPPS,  HARRIS,  see  John  M.  Dowling,  382,  C.  J. 
Karwoski,  385,  and  R.  Bruce  Szamier,  396 

RNA,  messenger,  role  in  sea  urchins,  372 
synthesis  in  cleaving  sea  urchin  embryos,  375 
tubulin,  tissue  specific  expression,  378 

ROBERTS,  SUSAN,  see  Cynthia  A.  Paden,  401 

Role  of  daily  light  period  and  intensity  in  photo- 
synthesis and  production  of  Zostera  marina 
L.  (eelgrass),  364 

Role  of  shoot  photosynthesis  in  root-rhizome  res- 
piration in  Zostera  marina  L.  (eelgrass),  368 

Role  of  the  germinal  vesicle  in  the  1-methylade- 
nine-induced  changes  in  protein  synthesis  in 
Asterias  oocytes,  The,  374 

Root-rhizome  respiration  in  Zostera,  368 

Ross,  D.  M.,  see  I.  D.  Lawn,  188 

ROWSE-EAGLE,  DEBRA,  CARL  A.  BOSWELL,  TECIA 
ULISSES  DE  CARVALHO,  AND  MIERCIO  PER- 
EIRA,  Comparison  of  labled  membrane  pro- 
teins and  nonpathogenic  South  American  try- 
panosomes,  394 

RUDERMAN,  JOAN,  see  Kristi  Wharton,  378 


Salicornia,  density  effects  on  growth  and  survival, 
365 

Salinity,  348 

Salinity  increases,  selection  for  halophilic  bacteria, 
369 

SALMON,  E.  D.,  M.  MCKEEL,  T.  S.  HAYS,  AND  C. 
RIEDER,  Rapid  rates  of  colchicine-  or  col- 
cemid-induced spindle  microtubule  disassem- 
bly in  vivo:  implications  for  the  mechanism 
of  microtubule  assembly,  361 

Salt  marsh,  wrack  accumulation  and  vegetation, 

368 

anoxic  decomposition,  370 
sediments  incubating  colonized  filter  paper,  37 1 


528 


INDEX   TO  VOLUME    163 


Salt  transport,  131 

SALZBERG,  B.  M.,  A.  L.  OBAID,  H.  SHIMIZU,  R.  K. 
ORKAND,  AND  D.  M.  SENSEMAN,  Does  the 
Schwann  cell  of  Loligo  act  as  a  potassium 
electrode?  Optical  studies  using  potentiomet- 
ric  probes,  390 

SALZBERG,  B.  M.,  see  A.  L.  Obaid,  388 

Sand  dollar,  sperm  movement  during  extraction, 
362 

SANGER,  JEAN  M.,  see  Mark  Bennett  Pochapin, 
376 

SANGER,  JOSEPH  W.,  see  Mark  Bennett  Pochapin. 
376 

Sarcomere,  329 

SARMA,  VIDYA,  see  Dennis  Goode,  358 

SATO,  EIMEI,  S.  J.  SEGAL,  AND  S.  S.  KOIDE,  Sperm 
agglutinating  factor  isolated  from  Spisula  oo- 
cytes,  376 

Scallops,  225 

SCHEIBLING,  R.  E.,  see  Stephen  A.  Watts,  348 

Schistosoma,  identification  of  antigens,  392 
membrane  labeling,  394 

Schistomiasis,  competitive  inhibition  test  for  di- 
agnosis of,  393 

SCHMIDT,  ANNE  W.,  see  David  Bodznick,  380 

SCHMIDT,  THOMAS  M.,  AND  RICHARD  W.  CAS- 
TENHOLZ,  The  effects  of  sulfide  on  cyanobac- 
terial  photosynthesis  in  marine  microbial  mats, 
368 

SCHNELL,  D.  J.,  T.  M.  PERNACK,  AND  H.  L.  KORN- 
BERG,  Effect  of  methyl  cv-D-glucoside  on  the 
growth  of  enteric  bacteria:  inhibition  and  es- 
cape from  inhibition,  403 

SCHNELL,  D.  J.,  see  H.  L.  Kornberg,  401 

SCHUEL,  H.,  E.  TRAEGER,  R.  SCHUEL,  J.  BOLDT, 
AND  M.  ALLIEGRO,  Indomethacin,  an  anti-in- 
flammatory drug,  promotes  polyspermy  in  sea 
urchins,  377 

SCHUEL,  HERBERT,  PRAMILA  DANDEKAR,  AND 
REGINA  SCHUEL,  Urea  parthenogenetically 
activates  the  cortical  reaction  and  elongation 
of  microvilli  in  eggs  of  the  sea  urchin,  Slron- 
gylocentrotus  purpuratus,  337 

SCHUEL,  REGINA,  see  Herbert  Schuel,  337,  377 

SCHUETZ,  ALLEN  W.,  see  Frank  J.  Longo,  453,  465 

Schwann  cell  as  potassium  electrode,  390 

Scyphozoa,  320 

Sea  anemone,  pedal  disc  release  in  Tealia  electrical 
conduction  system  in  Tealia,  188 

Searobin,  see  Prionotus 

Seasonal  changes  in  the  circadian  modulation  of 
sensitivity  of  the  Limuhis  lateral  eye,  380 

Seasonal  variation  in  the  flux  of  algal  pigments  to 
a  deep-water  site  in  the  Panama  Basin,  363 

Sea  star,  see  Asterias 

Sea  urchin,  eggs,  337 

role  of  maternal  mRNA,  372 
synthesis  of  RNA  and  tRNA  in  cleaving  em- 
bryos, 375 

polyspermy  promoted  by  Indomethacin,  377 
tissue-specific  expression  of  tubulin  RNAs  dur- 
ing development,  378 
protein  activation  of  sperm,  420 

Sediments,  oil  contaminated,  363 


SEGAL,  S.  J.,  see  Hideo  Mohri,  374,  and  Eimei 
Sato,  376 

Selection  and  properties  of  glucose  transport  mu- 
tants of  Vibrio  parahaemolyticus,  40 1 

Selection  for  moderately  halophilic  bacteria  by 
gradual  salinity  increases,  369 

Semilunar  spawning  cycle  in  a  Woods  Hole  pop- 
ulation of  Fundulus  heteroclitus,  369 

SENSEMAN,  D.  M.,  see  B.  M.  Salzberg,  390 

Sensory  lesion,  144 

SHIMIZU,  H.,  see  A.  L.  Obaid,  388,  and  B.  M.  Salz- 
berg, 390 

Shoot  photosynthesis  in  Zostera,  368 

Shrimp,  329 

SlEBENALLER,   JOSEPH    F.,    GEORGE    N.    SOMERO 

AND  RICHARD  L.  HAEDRICH,  Biochemical 
characteristics  of  macrourid  fishes  differing  in 
their  depths  of  distribution,  240 

SILVER,  WAYNE  L.,  AND  THOMAS  E.  FINGER,  Re- 
sponses from  spinally  innervated  chemorecep- 
tors  on  the  fin  rays  of  the  searobin,  Prionotus 
carol  inns,  390 

SILVER,  WAYNE  L.,  see  P.  D.  Prasada  Rao,  389 

SlLVERMAN,  EDWIN  K.  AND  JEAN  M.  HARTMAN, 
Wrack  accumulation  and  vegetation  structure 
in  Great  Sippewissett  Salt  Marsh,  368 

Skate,  retina,  382 

light-evoked  field  potentials  in  retina,  385 
rods,  postnatal  morphogenesis,  396 

Skeletal  muscle  of  the  frog,  276 

Skeleton,  ophiuroid,  431 

SLOBODA,  ROGER  D.,  see  William  D.  Cohen,  356, 
and  Anthony  C.  Breuer,  381 

SMITH,  DAVID  S.,  see  Gladys  Escalona  de  Motta, 
276 

SMITH,  ROBERT  D.,  WILLIAM  C.  DENNISON,  AND 
RANDALL  S.  ALBERTE,  Role  of  shoot  photo- 
synthesis in  root-rhizome  respiration  in  Zos- 
tera marina  L.  (eelgrass),  368 

SMITH,  S.  GREGORY,  see  Clifford  V.  Harding,  358 

Socci,  R.,  N.  CURTIS,  A.  FARMANFARMAIAN,  AND 
A.  ZWEIFACH,  Interactions  of  several  heavy 
metals  with  L-leucine  transport  in  the  intestine 
of  the  toadfish,  Opsanus  tan,  36 1 

So,  FREDERICK,  see  Frank  J.  Longo,  465 

Soman,  characterization  of  enzyme,  401 

Somatotopy  in  the  representation  of  the  pectoral 
fin  and  free  fin  rays  in  the  spinal  cord  of  the 
searobin,  Prionotus  carolinns,  154 

Somatotopy  within  the  medullary  electrosensory 
nucleus  of  the  skate.  Raja  erinacea,  380 

Some  membrane  structural  changes  accompanying 
morphogenetic  changes  in  Tetrahymena,  359 

SOMERO,  GEORGE  N.,  see  Joseph  F.  Siebenaller, 
240 

Spartina,  effect  of  nitrogen  on  respiration,  367 

Spawning,  semilunar  cycle  of  Fundulus,  369 

Spectroscopic  measurements  of  some  cytotoxic 
quinones,  399 

Sperm  agglutinating  factor  isolated  from  Spisula 
oocytes,  376 

Sperm  motility,  355,  492 

movement  during  extraction  with  Triton  X-100 
from  sand  dollar,  362 


INDEX   TO  VOLUME    163 


529 


aster  development  in  Asterias,  453 
Spicules  of  echinoplutei,  264 
Spinal  cord,  1 54 
Spinal  taste,  154 
Spirochaeta,  characterization  of  transport  systems, 

401 

Spisula,  regions  of  microtubule  assembly,  358 
meiosis  initiation  in  oocytes,  372 
sperm  agglutinating  factor,  376 
Sponge  aggregation,  stimulus/response  coupling 

371 

Spore-forming  bacteria,  germination  properties 
370 

Squid,  analysis  of  membranous  organelle  transport 

in  axons  and  axoplasm,  382 
use  and  construction  of  carbon  fiber  electrode 

in  axons,  386 
calcium-dependent  potassium  current  in  presyn- 

aptic  nerve  terminals,  397 
giant  axons,  400 

salivary  gland  enzyme  characterization,  401 
axons,  402 
giant  synapse,  perfusion  of  stellate  ganglion 

through  blood  supply,  403 

Stage-specific  gene  expression  in  Plasmodium  gal- 
linaceum,  391 

STANLEY,  E.  F.  AND  W.  J.  ADELMAN,  JR.,  Perfu- 
sion of  the  squid  stellate  ganglion  through  its 
blood  supply:  implications  for  morphological 
and  physiological  studies  of  the  squid  giant 
synapse,  403 

Stenotomm,  infrastructure  and  distribution  of  lat- 
eral line  and  ocular-associated  structures,  358 

STEPHENS,  LAURIE  E.,  A  study  of  the  heat  shock 
response  in  early  embryos  of  Spisula  so/idis- 
sima,  377 

STEPHENS,  PHILIP  J.,  see  Kathleen  O'Connor,  329 

Stimulus/response  coupling  in  sponge  aggregation: 
evidence  for  calcium  as  an  intracellular  mes- 
senger, 371 

STODDARD,  JEFFREY  J.,  Semilunar  spawning  cycle 
in  a  Woods  Hole  population  of  Fundulus  het- 
eroclitus,  369 

Sulfide,  effect  on  cyanobacterial  photosynthesis, 
368 

SUNDELL,  CYNTHIA  L.,  AND  TIM  OTTER,  Vigorous 
movement  of  sand  dollar  sperm  during  ex- 
traction with  Triton  X-100,  362 

Surface  labeling  of  Trypanosoma  cruzi,  391 

SUSAN,  STANLEY  R.,  see  Clifford  V.  Harding,  358 

Suspension  feeding,  197 

Synchronous  flashing  in  Photimts,  398 

Synthesis  and  release  of  3H-octopamine  from  the 
cardiac  ganglion  of  Limulus  polyphe mus,  387 

Synthesis  of  5S  RNA  and  tRNA  in  cleaving  sea 
urchin  embryos:  effect  of  altering  cell  inter- 
actions, 375 

SZAMIER,  R.  BRUCE,  HARRIS  RIPPS,  AND  DOUG- 
LAS TAATJES,  Evidence  for  postnatal  mor- 
phogenesis of  skate  rods,  396 

SZAMIER,  R.  B.,  see  C.  J.  Kanvoski,  385 

SZENT-GYORGYI,  ALBERT,  see  Peter  R.  C.  Has- 
coyne,  399 


TAATJES,  D.  J.,  see  C.  J.  Karwoski,  385,  and  R. 
Bruce  Szamier,  396 

Tanganella  appendiculata,  n.  sp.,  172 

TASAKI,  L,  see  R.  M.  Gould,  400 

TAYLOR,  KEVIN,  see  David  Landowne,  386 

Tealia,  see  sea  anemone,  1 88 

Teleost,  131,  marine,  see  Stenotomus 

TELFORD,  MALCOLM,  see  Gergard  Pohle,  2 1 1 

Temperature,  348 

Temperature-induced  disassembly  of  isolated  mar- 
ginal bands  and  reassembly  of  marginal  band 
tubulin,  356 

Tissue-specific  expression  of  tubulin  RNAs  during 
sea  urchin  development,  378 

TRAEGER,  E.,  see  H.  Schuel,  377 

Transduction  and  voltage-dependent  currents  of 
statocyst  hair  cells  in  Hermissenda,  399 

Transepithelial  permeability  in  ocular  lens  epithe- 
lium, 360 

Transport  ATPases,  108 

Trigonometric  nearest  neighbor  analysis  of  the 
neuroplasmic  lattice  arrays  in  axons,  379 

TROLL,  W.,  see  C.  Aranow,  355,  and  James  P. 
Quigley,  402 

Trypanosoma,  surface  labeling,  391 

Trypanosomes,  comparison  of  labeled  membrane 
proteins,  394 

Turbellarid  infection  in  Limulus  gill,  392 

Tubulin  gene  expression,  control  during  Leish- 
mania  transformation,  39 1 

Tubulin  reassembly,  356 

Tuna,  356 

Two  cell  volume  regulatory  systems  in  the  Limulus 
myocardium:  an  interaction  of  ions  and  qua- 
ternary ammonium  compounds,  504 


u 

Uca,  251,  301 

ULISSES  DE  CARVALHO,  TECIA  MARIA  AND  MIER- 
cio  PEREIRA,  Surface  labeling  of  Trypano- 
soma cruzi,  39 1 

ULISSES  DE  CARVALHO,  TECIA,  see  Debra  Rowse- 
Eagle,  394 

Uptake  and  utilization  of  L-alanine  by  10  species 
of  bivalve  molluscs,  360 

Urea  activated  eggs,  337 

Urea  parthenogenetically  activates  the  cortical  re- 
action and  elongation  of  microvilli  in  eggs  of 
the  sea  urchin,  Strongvlocentrotus  purpuratus, 
337 


VALIELA,  IVAN,  see  Osiris  Boutros,  362 

Vanadate,  395 

Vegetation  structure  in  salt  marsh,  368 

VENTOSA,  A.,  J.  S.  POINDEXTER,  AND  W.  S.  REZ- 
NIKOFF,  Selection  for  moderately  halophilic 
bacteria  by  gradual  salinity  increases,  369 


530 


INDEX  TO   VOLUME    163 


VERAKALASA,  PACHARA,  AND  TOM  HUMPHREYS, 
A  low  molecular  weight  subunit  of  the  aggre- 
gation factor  complex  of  Microciona  prolifcra 
that  stoichiometrically  binds  to  and  inhibits 
the  intact  aggregation  factor,  378 

Vestibular  efferent  neurons,  central  organization 
of,  384 

I'ihrio,  glucose  transport  mutants,  401 

Victorella  pseudoarachnidia,  n.  sp.,  172 

Vigorous  movement  of  sand  collar  sperm  during 
extraction  with  Triton  X-100,  362 

Visual  cortex,  optical  monitoring  of  evoked  activ- 
ity, 389 

Vitellaria,  431 

Vitelline  layer,  337 

Vitellogenesis  in  the  hepatopancreas  and  ovaries 
of  Carcinus  maenas,  375 

Voltage-dependent  currents  of  Hermissenda  sta- 
tocyst  hair  cells,  399 

Volume  control  mechanisms,  invertebrate  cell,  405 

VOSSHALL,  LESLIE,  see  Philip  Dunham,  420 


w 

WALCH,  MARIANNE,  PAUL  J.  BOYLE,  AND  RALPH 
MITCHELL,  Comparative   microbiology  of 
metal  surfaces  in  sea  water,  403 
WALDRON,  WILLIAM,  see  Seymour  Zigman,  404 
WALTZ,  R.  B.,  see  W.  J.  Adelman,  379 
WARREN,  MARY  KIM  AND  SIDNEY  K.  PIERCE, 
Two  cell  volume  regulatory  systems  in  the 
Limitlus  myocardium:  an  interaction  of  ions 
and  quaternary  ammonium  compounds,  504 
WATTS,  STEPHEN  A.,  R.  E.  SCHEIBLING,  ADAM  G. 
MARSH,  AND  JAMES  B.  MCCLINTOCK,  Effect 
of  temperature  and  salinity  on  larval  devel- 
opment of  sibling  species  of  Echinaster  (Echi- 
nodermata:  Asteroidea)and  their  hybrids,  348 
WEISSBURG,  MARC,  ALLYSON  SENIE,  GEORGE 
KOWALLIS,  AND  JOSEF  TREGGOR,  The  de- 
velopment and  geomorphology  of  Great  Sip- 
pewisset  Marsh  (Falmouth,  MA):  the  Redfield 
model  revisited,  370 


WEISSMANN,  GERALD,  see  Cathleen  Anderson, 
371,  and  Philip  Dunham,  420 

WHARTON,  KRISTI,  GLENN  MERLINO,  RUDOLF 
RAFF,  AND  JOAN  RUDERMAN,  Tissue-specific 
expression  of  tubulin  RNAs  during  sea  urchin 
development,  378 

WIER,  P.,  A.  KEYNANA,  AND  H.  O.  HALVORSON, 
Germination  properties  of  a  marine  spore- 
forming  bacterium,  370 

WIGLEY,  ROLAND  L.,  see  Don  Maurer,  477 

WILLEY,  JOANNE,  AND  ROBERT  W.  HOWARTH,  A 
comparative  study  of  anoxic  decomposition 
in  salt  and  freshwater  marshes,  370 

Winter  flounder,  see  Pseudopleuronectes 

WIRTH,  DYANN,  see  Jay  Bangs,  391,  Marie-France 
Delauw,  391,  and  Pamela  Langer,  393 

WOGRIN,  N.,  J.  S.  POINDEXTER,  AND  E.  P.  GREEN- 
BERG,  Microbial  colonization  of  filter  paper 
incubated  in  salt  marsh  sediments  as  observed 
by  scanning  electron  microscopy,  371 

WOODRUFF,  R.  I.,  D.  A.  LUTZ,  AND  S.  INOUE, 
Lucifer  yellow  CH  as  a  non-intrusive,  in  vivo 
fluorescent  probe  for  physiological  studies 
during  early  development,  379 

Wrack  accumulation  and  vegetation  structure  in 
Great  Sippewissett  Salt  Marsh.  368 


YOUNGBLOM,  JIM,  see  Tom  Evans,  372 
Young's  modulus  of  echinoderm  calcite,  264 


ZEICHNER,  STEVEN,  see  Jay  Bangs,  391 

ZlLBERSTEIN,    DAN,    PAUL    JOHNSON,    MlERCIO 

PEREIRA  AND  DON  HARN,  Membrane  label- 
ing of  protective  antigens  of  schistosomula  of 
Schistosoma  mansoni,  394 

Zonulae  occludentes  and  transepethilial  perme- 
ability in  the  ocular  lens  epithelium,  360 

Zostera,  see  eelgrass 

ZWEIFACH,  A.,  see  R.  Socci,  361 


Continued  from  Cover  Two 

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CONTENTS 


Invited  article: 

PIERCE,  SIDNEY  K. 

Invertebrate  cell  volume  control  mechanisms:  a  coordinated  use  of  in- 
tracellular  amino  acids  and  inorganic  ions  as  osmotic  solute     405 


DUNHAM,  PHILIP,  LEONARD  NELSON,  LESLIE  VOSSHALL,  AND  GERALD 

WEISSMAN 

Effects  of  enzymatic  and  nonenzymatic  proteins  on  Arbacia  spermato- 
zoa: reactivation  of  aged  sperm  and  the  induction  of  polyspermy  ....    420 


HENDLER,  GORDON 

An  echinoderm  vitellaria  with  a  bilateral  larval  skeleton:  evidence  for 
the  evolution  of  ophiuroid  vitellariae  from  ophioplutei    .  .  .  1 431 


KANUNGO,  K. 

In  vitro  studies  on  the  effects  of  cell-free  coelomic  fluid,  calcium,  and/ 
or  magnesium  on  clumping  of  coelomocytes  of  the  sea  star  Asterias 
forbesi  (Echinodermata:  Asteroidea)  .i  .».  .\ 438 


LONGO,  FRANK  J.,  AND  ALLEN  W.  SCHUETZ 

Male  pronuclear  development  in  starfish  oocytes  treated  with  1-meth- 
yladenine  .  .  .>vv; . ._.'.  ^  i'V--*  *?••«! 453 


LONGO,  FRANK  J.,  FREDERICK  So,  AND  ALLEN  W.  SCHUETZ 

Meiotic  maturation  and  the  cortical  granule  reaction  in  starfish  eggs    465 

A./  «5  v_~     '     J      ^  N.  \_       -v '*"  •"'  -' 

MAURER,  DON,  AND  ROLAND  L.  WIGLEY 

Distribution  and  ecology  of  mysids  in  Cape  Cod  Bay,  Massachusetts    477 

x  •   '      •    " J  -     •  -    *N"  i • 


NELSON,  LEONARD 

Membrane-stabilizing  and  calcium-blocking  agents  affect  Arbacia  sperm 
motility    J?:fefc$!jj; 492 


WARREN,  MARY  KIM,  AND  SIDNEY  K.  PIERCE 

Two  cell  volume  regulatory  systems  in  the  Limulus  myocardium:  an 
interaction  of  ions  and  quaternary  ammonium  compounds    504 


INDEX  TO  VOLUME  163  517